0 1984 hv The American Society of Biological Chemists, Inc. THE JOURNAL OP BIOLOGICAL CHEMISTRY Vol. 259, No. 2, Issue of January 25, pp. 1051-1055,1984 Printed in U.S.A.

Proteins of the Bacillus stearothermophilus Ribosome THE AMINO ACID SEQUENCES OF PROTEINS S5 AND L30*

(Received for publication, July 28, 1982)

The amino acid sequences of ribosomal proteins S5 and L30 from Bacillus stearothermophilus have been determined. These proteins have recently been crys- tallized in our institute. Sequence data were obtained by manual sequencing of peptides derived from cyan- ogen bromide cleavage and digestion with trypsin and chymotrypsin or thermolysin. Proteins 55 and L30 contain 166 and 62 amino acid residues and have cal- culated M, values of 17,628 and 7,053, respectively. Comparison of the sequences with those of the homol- ogous proteins from Escherichia coli shows 55% iden- tical residues for S5 and 53% for L30. For both pro- teins, the distribution of conserved and substituted regions is not uniform throughout the molecule. Sec- ondary structure predictions were carried out for the B. stearotherrnophilus proteins. Comparison with the results for the homologous E. coli proteins indicated similar secondary structural order for the molecules from the two species.

The study of the structure of ribosomal proteins is now progressing toward the elucidation of the tertiary structure by x-ray crystallography. As described,’ it is known that proteins from thermophilic bacteria possess more stable struc- tures and might be better candidates for physicochemical studies in solution and for crystallization than those from mesophiles. Therefore, ribosomal proteins from the thermo- philic organism Bacillus stearotherrnophilus have been iso- lated using a mild extraction procedure’ and subjected to crystallization experiments. Four ribosomal proteins (S5, L6, L9, and L30) have been crystallized so far in our institute (2- 4), and x-ray crystallographic determinations of these struc- tures are now in progress.

Simultaneously, amino acid sequence analyses have been carried out on proteins isolated in this way to correlate the proteins with their homologues from Escherichia coli, and also to facilitate the tertiary structure analysis of the proteins which have been crystallized. Since a significant amount of sequence data is now available for B. stearothermophilus proteins (4, 5) ,2 Appelt and Dijk’ proposed a new nomencla- ture for B. stearothermophilus ribosomal proteins. These are now numbered as their E. coli homologue as established by amino acid sequence (complete or partial), since this facili- tates the integration of results for proteins from the two organisms. I have adhered to this numbering system through- out this paper.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ K. Appelt and J. Dijk, manuscript in preparation. ’ M. Kimura, unpublished results.

Data on the crystals and a more general discussion of S5 and L30 can be found in Ref. 3 and references cited therein. I here present the amino acid sequences of S5 and L30 and a comparison with those of the homologous proteins from the E. coli ribosome. The NH,-terminal sequence of S5 has been reported previously (6).

EXPERIMENTAL PROCEDURES

Most of the materials and methods used in sequencing S5 and L30 have been described in Ref. 7. Additional information may be found in Ref. 8. All studies were performed on proteins extracted from B. stearothermophilus strain NCA 1503 purchased from the Centre for Applied Microbiology and Research, Porton, England.

RESULTS~

The Amino Acid Sequence of S5”The complete primary structure of S5 is shown in Fig. 1. This structure was deter- mined by obtaining amino acid sequences for two cyanogen bromide peptides CBI (position 2-136) and CBII (position 137-166), which were separated on Sephadex G-75 (super- fine). Amino acid compositions (Table I) and NHa-terminal sequences of these peptides suggested that the alignment of peptides is CBI-CBII.

Both peptides were then subjected to sequence analysis. As shown in Fig. 1, while the complete sequence of CBII was determined by manual solid phase sequencing, only the NH2- terminal sequence was obtained for CBI. Therefore, this pep- tide was further digested with trypsin and chymotrypsin, and the resulting peptides were isolated as shown in Fig. 2. Amino acid compositions of these peptides are given in Tables I1 and 111. All tryptic and chymotryptic peptides except T13 (position 70-112) and T14 (position 113-126) were isolated by peptide mapping and sequenced by the 4-N,N-dimethylaminoazoben- zene 4’-isothiocyanate/phenyl isothiocyanate double coupling method. The isolation of T13 and T14 as well as TM1, which provided proof for the alignment of peptides CBI and CBII, was carried out by gel filtration on Sephadex G-50 (superfine) followed by mapping of tryptic peptides from S5. Amino acid sequences of these peptides were determined by manual solid phase sequencing.

In this way, the complete amino acid sequence of S5 was established as shown in Fig. 1. S5 consists of 166 residues, and from this composition an M , of 17,628 was calculated. The amino acid composition derived from the sequence agrees

Portions of this paper (Figs. 2 and 4 and Tables I-VI) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. The abbreviation used is: Hse, homoserine. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-2044, cite the authors, and in- clude a check or money order for $4.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

1051

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1052 Amino Acid Sequences of Ribosomal Proteins S5 and L30 10 20 30

Met-Aq-Arg-I le-Asn-Pro-Awl-Lys-Leu-Glu-Leu-Glu-Glu-Arg-Val-Val-Ala-Val-Asn-Arg-Val-Ala-Lys-Val-Val-Ly~-G~y-G~y-Arg-Aq

C NBr

TRY 11 . , T 2 , , 7 3 I L ” + 15 16 7 7 ” ” ” ” ” ” ” ” ~ ” ” ~ 7 7 -77

CBI ” ” ” ”7” ”

CHYM , . c 2 , . c3 , , c 4 7”””””””” 77-7” 77-17

TRY - b 18 , . T9 , ,T10

”7” ” ” ” ””””-7 77”””

(HYM + & 8 c6 , , CI ” ” 7 ” 7 ” ” ” 7 7 ” ” ” - 7 ” ”

70 80 90 A q - L y s - A l a - l l e - G l u - A s p - A l a - L y s - L y s - A s n - L e u - I l e - G l u - V a l - P r o - I l e - V a l - G l y - T h r - T h r - I l e - P r o - H i s - G l u - V a l - l l e - G l y - H i s - P h e - G l y

CNBr

111 112 TRY - Y I 111 713

1 - r 7 7”” ) c L ) ) ) C C L C c C e ” ) L L C c L

CHYM , , C B c9 ” 77 ” ”777 77””

I C c

100 110 120 A l a - G l y - G l u - l l e - I l e - L e u - L y s - P r o - A l a - S e r - G l u - G l y - T h r - G l y - V a l - l l e - A l a - G l y - G l y - P r o - A l a - A q - A l a - V a l - L e u - G l u - L e u - A l a - G l y - l l e

CNBr

130 140 150 Ser -Asp- I le -Leu-Ser -Lys-Ser - I l e -G ly -Ser -Asn-Thr -Pro- I le -Asn-Met -Va l -Aq-Ala -Thr -Phe-Asp-Gly -Leu-Lys-Gln -Leu-Lys-Aq-Ala -

160

Glu-Asp-Va l -A la -Lys-Leu-Aq-Gly -Lys-Thr -Va l -G lu-Glu-Leu-Leu-Gly

CNBr ” e c c c c c c ~ c c c c c ~ ~

FIG. 1. The amino acid sequence of 55 from B. stearothermophilus. Sequence data on individual peptides are indicated as follows: -, sequenced by 4-N,N-dimethylaminoazobenzene 4-isothiocyanate/phenyl isothiocyanate double coupling method; b, sequenced by solid phase procedure using 1-ethyl-3-dimethylaminopropyl carbodiimide in the coupling reaction; D, sequenced by solid phase procedure by p-phenylenediisothiocyanate method. CNBr, TRY, and CHYM indicate peptides derived from cleavage with cyanogen bromide, trypsin, and chymotrypsin, respectively.

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Amino Acid Sequences of Ribosomal Proteins S5 and L30 1053

10 20 30 A~o-Lys-Lys-Leu-A lo- l le -Thr -Leu-Thr -Arg-Ser -Va l - l le -G ly -Aq-Pro-Glu-Asp-Gln-Arg- l le -Thr -Vo l -Aq-Thr -Leu-Gly -Leu-Arg-Lys-Met -

TRY

CNBr

TH

TRY

CNBr

11 72 1 3 “I

TS 16 12 17 7 7 ” 7 7 7 7 7 7 7 7 ~ 7 7 7 7 ~ ” ” ” ” ” 7

, , T L 4-1 I H C

CB1 + ”777””77777

,CBIThl , CBITh2, ,CBITh3, .CBTh4 , , CBlThS , , CBITh6 CB11h7 CBITh8 I 1 - 1 ” 7 7 ” ” ” ” 1 7 7 1 7 1 7 7

LO 50 60 H i s - G l n - T h r - V o l - V o l - H i s - A s n - A s p - A s n - P r o - A l o - l l e - A q - G l y - M e t - l l e - A s n - L y s - V a l - A l a - H i s - L e u - V a l - L y s - V a l - L y s - G l u - l l e - G l u ( G I ~ ~ G l ~ )

, , T8 , . T 9 , I T10 4

7”77 ”17 ”7 ”777-7 71 -17 ””-”

CBII , , CBm 4 7777-7777 ”7 ”7 ”7 ” ”7

FIG. 3. The amino acid sequence of L30 from B. stearothermophilus. Sequence data on individual peptides are indicated in the same way as in Fig. 1. TRY, CNBr, and TH indicate peptides derived from cleavage with trypsin, cyanogen bromide, and thermolysin, respectively.

The Amino Acid Sequence of U0-The amino acid sequence of L30 is shown in Fig. 3. The proposed sequence was deduced as follows. L30 was first digested with trypsin, and the pep- tides were isolated by mapping as indicated in Fig. 4. Amino acid compositions of these peptides are given in Table V. Amino acid sequences of the peptides were determined as shown in Fig. 3. Alignment of tryptic peptides was obtained from amino acid sequences or compositions of cyanogen bro- mide peptides, as well as thermolytic peptides derived there- from. Cyanogen bromide cleavage of L30 gave three peptides, CBI, CBII, and CBIII, which were separated by gel filtration on Sephadex G-50 (superfine). Amino acid compositions and sequences of these peptides are give in Table VI and Fig. 3, respectively. Furthermore, peptide CBI was digested with thermolysin, and the resulting peptides were isolated and sequenced. From these results, the amino acid sequence of L30 was determined and is shown in Fig. 3. L30 contains 62 amino acid residues, and an M , of 7053 was calculated from the composition. The composition derived from the sequence and that calculated from amino acid analysis of acid hydrol- ysates of whole protein agree well (Table IV).

DISCUSSION

The complete amino acid sequence of S5 as shown in Fig. 1 was obtained from a single preparation of the protein. Also a partial sequence (96 out of the total 166 positions) was obtained from a small amount of S5 isolated from a different batch of B. stearothermophilus strain NCA 1503. In this partial sequence, eight differences were observed, namely Asn- 5 + Asp, Asn-7 + Ser, Ala-17 + Thr, Glu-58 - Asp, His-88 + Lys, Glu-93 + Lys, Ser-100 3 Val, and Ala-150 + Val. This significant number of differences between the two pro- tein preparations cannot be explained in a satisfactory man- ner at present. However, three different observations have some relevance to this problem.

First, in the determination of partial or complete sequences of other ribosomal proteins from the same two batches of bacteria, no differences have been found.2 Secondly, Higo et al. (6) determined the NH2-terminal sequence of S5 from B. stearothermophilus strain 799 and reported an aspartic acid at position 5. This is in contrast with the asparagine reported in the complete sequence (Fig. 1) but agrees with the aspartic acid found in the partial sequence determination. However, the remainder of the 30 NH2-terminal residues of strain 799 agrees perfectly with the complete sequence of NCA 1503.

Third, two reports on the NH2-terminal sequence of S5 from Bacillus subtilis strain ATCC 6633, which is closely homolo- gous to S5 from B. stearothermophilus, have appeared (6, 9). The two sequences are identical except for a discrepancy in position 7 where an Asn versus Ser change occurs. The same discrepancy was observed in the two B. stearothermophilus sequences presented here. In the B. subtilis sequence, Asp is found in position 5 and Thr in position 17, in agreement with the partial sequence.

For E. coli, the sequences of S5 from strain B and strain K have been compared and appear only to differ in the presence of glutamic acid or alanine at position 150 in the two respec- tive strains (10). Several mutations in E. coli S5 related to antibiotic resistance in the organism have been reported and are summarized in Ref. 10. The mutations cluster in two regions of the sequence, but none of these correlate with the positions where we observe changes in the B. stearothermo- philus S5 sequence between the two preparations.

Studies of this protein are being continued, and I hope these will clarify the significance of the differences between the complete and the partial sequences.

Spectinomycin-resistant mutants of B. stearothermophilus have also been produced in this institute, and S5 is being extracted from these mutant b a ~ t e r i a . ~ We will determine the sequences of these mutants, and accompanying studies will be made of the tertiary structure in the crystal. This will hopefully lead to an understanding of the role of S5 in antibiotic resistance at the molecular level.

I t is unclear why peptide T1, Arg-Ile-Asn-Pro-Asn (position 3-7), was produced by tryptic digestion of S5. It seems un- likely that there is sufficient chymotrypsin contaminating the trypsin to carry out this specific cleavage between asparagine and lysine.

In the sequence determination of L30, when peptide TI0 (position 57-62) was sequenced by the solid phase sequencing method, a good deal of the previous amino acid was carried over into the current amino acid position. This meant that for the last 2 amino acid residues it was not possible to distinguish between glutamic acid and glutamine although both degradations indicated glutamic acid. The possibility that these glutamic acids came from the glutamic acid at position 60 cannot be absolutely excluded. These residues are included in parentheses (in Fig. 3) as glutamic acid at present,

K. Isono and J. Dijk, unpublished results.

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1054 Amino Acid Sequences of Ribosomal Proteins S5 and L30

and the final resolution of these two positions must await further material.

The amino acid sequences of S5 and L30 are compared with those of S5 (10) and L30 (11) from E. coli, respectively, in Fig. 5 . B. stearothermophilus S5 has 1 extra residue (methio- nine) at the NH, terminus and lacks the carboxyl-terminal residue (lysine in E. coli) when the residues are aligned so as to optimize the similarity. There are identical residues at 90 positions in the two sequences, that is at 55% of the residues compared, and at a considerable number of the other positions there are chemically similar residues. B. stearothermophilus

0 1 1 -

20

NYNIYMYI

I -1

M___ I

- t t

"1 Nm 1 1 m 1M

m 100

_m_

t rrm_

t -

f f - -."--"I 1 I

120

BSS P A~A-~:L;D;:S O ~ ; [ S ! S I UN T ~ M ~ F ~ K

~ S S A W R ~ V L E ~ ~ A G ~ V ~ ~ N ~ V ~ L A K L Y G S ~ N P I N V V ~ . l I O G L E

110

120 110

-1 -1 ______1

160

1.50

t -

1 -

BL 30

t t)3

EL30

EL IO

FIG. 5. Comparison of the primary structures and predicted secondary structures of two homologous proteins from B. stearothermophilus and E. coli. a, comparison of the proteins S5; b, comparison of the proteins L30. Identical residues are enclosed in solid boxes. Conservative replacements (Glu, Asp, Lys, Arg; Ile, Leu; Ile/Leu, Val and Ser, Thr) are enclosed in broken-line boxes. The symbols used for secondary structure features are: cpu , a-helix; w , $-structure; T, first residue in &turn.

L30 has an extra 4 residues at the carboxyl terminus when compared with its E. coli homologue. There are identical residues at 31 positions in the two proteins, 53% of the total number.

Comparing the distribution of acidic and basic amino acid residues in the L30 proteins from the two organisms, there are some differences. There is a cluster of arginine and lysine residues in the COOH-terminal region (position 44-57) of B. stearothermophilus L30, but only 1 lysine and 1 arginine in the corresponding region of the E. coli protein. There are acidic amino acid residues, glutamic and aspartic acid, at positions 17 and 18, respectively, in B. stearothermophilus L30, but no acidic amino acid residues in region 1-35 of the E. coli sequence. In contrast, for S5, there appears to be a similar distribution of acidic, basic, and, indeed, aromatic residues in the sequences from the two organisms.

Furthermore, the degree of similarity between the species is not constant throughout the two pairs of sequences. For S5, there are highly conserved sequences in positions 17-57 and 98-144, with 73 and 68% identical residues, respectively. In contrast, the NH2-terminal 16 residues and those from position 75-97 are quite dissimilar. Similarly for L30, there is a much higher degree of homology in residues 1-15 and 41- 58 than in the central part (position 16-40). The identification of highly conserved regions in the sequence will greatly help the study of the functionally important sites in these ribo- somal proteins, particularly with regard to the information available from the x-ray structure.

Fig. 5 also shows a comparison of the secondary structures predicted by the method of Chou and Fasman (12). Perhaps not surprisingly in view of the high degree of homology between the pairs of proteins (>50%), there is a good agree- ment between the secondary structures predicted for the proteins from B. stearothermophilus and E. coli. The validity of these predictions will be tested by the elucidation of the x- ray structures of S5 and L30 from B. stearothermophilus.

Acknowledgments-I thank Dr. H. G. Wittmann for his continuous interest and encouragement and Drs. J. Dijk and K. Wilson for helpful discussion. The proteins S5 and L30 were supplied by Drs. J. Dijk and K. Appelt.

REFERENCES 1. Appelt, K., Dijk, J., and Epp. 0. (1979) FEBS Lett. 103,66-70 2. Appelt, K., Dijk, J., Reinhardt, R., Sanhuesa, S., White, S. W.,

Wilson, K. S., and Yonath, A. (1981) J. Biol. Chem. 256 ,

3. Appelt, K., White, S. W., and Wilson, K. S. (1983) J. Biol. Chem.

4. Kimura, M., Dijk, J., and Heiland, I. (1980) FEBS Lett. 121,

5. Kimura, M., Rawlings, N., and Appelt, K. (1981) FEBS Lett.

6. Higo, K.-I., Itoh, T., Kumazaki, T., and Osawa, S. (1980) in Genetics and Evolution of R N A Polymerase, t -RNA and Ribo- somes (Osawa, S., Ozeki, H., Vchida, H., and Yura, T., eds) pp. 655-666, University of Tokyo Press, Tokyo

7. Kimura, M., and Wilson, K. S. (1983) J. Bid. Chem. 258,4007- 401 1

8. Wittmann-Liebold,, B., and Lehmann, A. (1980) in Methods in Peptide and Protein Sequence Analysis (Birr, C., ed) pp. 49-72, Elsevier/North-Holland Biomedical Press, Amsterdam

9. Higo, K.-I., Itoh, T., and Osawa, S. (1982) Mol. Cen. Genet. 185,

10. Wittmann-Liebold, B., and Greuer, B. (1978) FEBS Lett. 9 5 ,

11. Ritter, E., and Wittmann-Liebold, B. (1975) FEBS Lett. 60,153-

12. Chou, P. Y., and Fasman, G. D. (1978) Adu. Enzymol. Relat. Areas

11787-11790

258,13328-13330

323-326

136,58-64

205-206

91-98

155

Mol. Biol. 47,45-148

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1055

I bl

0 0 0 0

0

Table I: Ammlno acld composltrons of CNBr peptrdes from s5

Peptlde C B I CBll

p s r t r o n s 2 - 136 137 - 1 6 6

T a b l e IV: ammo acld cornpasltlons of s 5 and 130

9 . 5 3 1 1 0 1 4 .8815)

1 3 . 4 0 1 1 2 1 5 . 5 9 1 6 )

1 7 . 1 0 1 1 7 l 6 .62171

13 .341131 15 .381161 12 .731141

9 .26191

2 .00121 1 .84121

4 .00141

2 .9613) 2.75133 2 .7313)

4 . 7 1 151

0 .8511)

4.30141 3 .58131

5 7 7 6

14 2 7

20 16 19 2

14 74 0 4 3

14 1 2 0 0

11 .51

6 .26

1 6 . 0 1 4 .63

18 .68 7 .36

1 6 . 1 0 1 8 . 4 5

1 . 6 7

1 3 . 6 8 1 2 . 1 2

0 3 . 5 0 3 . 1 6

1 4 . 6 2 1 2 . 6 7

0 0

2 3 5 1 5 2 2 1 4 7 2 6 5 0 0 3 6 6 0 0

5 . 0 5

4.45

6 . 2 5 1 .41

2 .09

4 .70 3 .40

6 . 2 8

5 .48 1 . 9 8

6 .06

2 . 9 7 6 . 3 5 6 . 0 0 0 0

3.00131 3 . 0 2 1 3 ) 9 .401101 8 . 9 1 I 9 1

flil

Tab le : AmlnD dcld composltians of trYPrlC peptides of peptide CB1 from 5 5

POSltlOnS 3-7 8-14 15-20 21-23 24-26 27-29 30-32 33-42

Peptrde TI T2 T3 T4 T5 T6 T7 ~8

Asx 1.7712; C Y S

Thr 1 .28111 1 . 0 0 1 1 ~

w: Amlno acrd conposlrrons of tryptic peprldes of L30

P051t10nS 1-2 3 .30 4-10 11-20 21-24 25-29 31-44 Peptide T1 T2 TI TI T5 T6 T7

9sx 1 . 0 0 1 1 1 1.00131 r h r ser

2.12121 0 . 7 0 1 , )

1.00111 0 .78111 0 .96111 . ._ ~

Le" Ile 1.00111

TYr Phe H l r LYS 0.92111

1 . 1 7 1 1 )

R r g 1.02111 1 .00111 1 .05111 1 .13111 1 .79121 0 .86111

Peptlde T9 T 1 0 T i l T 1 2 T l 3 T I 4 ~ 1 5

PosltiOnS 43-52 53-61 62.69 63-68 10-112 113-126 127-136

1.89121 1 .00111 0.88171

0 . 9 1 1 1 1 1 . 0 0 1 1 ~

G l X PTO

2 .31 i21 1 .27111 1 .41111 1 .30111 0 .86111 1 .01111

0 .96111 1 .80121

0 .89111 1 .23111 Val 0.74111 1 . 0 7 1 1 1 Met n L"',,

G l X 2 . 3 1 i 2 1 PTO

1.27111

0 .86111 1 .41111 1 .30111

1 .23111 0 .96111 0 .74111 1 . 0 7 1 1 1 1.80121

0 .60111 0 .91111

0 .89111 1 .01111

Val net I le 1 .00111 0 .7611) 0 .75111 Leu w r

1 . 9 0 1 2 1 1 . 8 3 1 2 ) I l e Leu w r

1 .00111 0 .7611) 0 .75111 1 . 9 0 1 2 1 1 . 8 3 1 2 )

"_"" , . , 0.91111

Phe ~ 1 .

CY 3 Asx 0 . 7 8 1 1 ) 0 . 9 2 1 1 1 1 .0011) 1 .13111 2.09121 T h r 0 . 8 1 1 1 ) Ser

2.54131 1 .00111

G l X Pro

0 .95111 1.48121 1 .67121 3 . 0 0 1 3 1 1 . 2 9 1 1 1 4 . 2 5 1 4 1 1 . 2 3 1 1 1 0 .78111

Gly 4.20141 4.36141 0.8011l 7 .72181 1 .30111 1 .57111

A l a 1.66 121 V a l 1.08111 1 .24111

1 .94121 3 .89141 2 .00121

Met 3 . 2 6 1 4 ) 1 . 5 1 1 1 1

Le" *.._ 1.93121 2 .77131 11e 1.27111 1 .OOl l l 5 .30171 1 .72121 2 .12121

Hse I 1 1

His. Lyr 1.00111 1 .00111 Arg

1.80121

0 .93111 2 .30121 1 . 1 1 1 1 1 1 .00111 1.01111

Peptlde T8 T9 T1O

AISX 1.12111

POsltionS 4 5 - 4 9 50-55 56-62

Thr

3.85141

HLS 0 . 9 9 1 1 ) Phe 0.9211)

Lys 1.07111 Arg 1.2511)

.I= 0 . 8 0 1 1 ~ 1 . 5 0 1 2 1

0 . 7 8 1 1 1 1.0011) 0.88111 0 . 9 5 1 1 1 0 . 8 8 1 1 )

0 .87111 1.01111 2 .12121 1 .00111

0 .88111 l.OOl11 0.86111

1.00111

H1s LYS 0 . 8 7 1 1 1 0 . 8 l l l I 0 . 1 2 1 1 ~ i v g

0.79111

CYS A I x 2 .18121 1 .00111 Th?

CYS AIX 2 . 1 8 1 2 1 1.00111 2 .16121 2 .31121 Thr

G l X Se r

1.11111

Pro 0.68111 3.98141

A l a Gly

0.90111 2.05121 3 .34131 2 .07121

0 .87111 1 .04111 V a l He f

0.97111 4.00141 2 .4313 ; 2 .52131 3 .25141 1 .11111

I l e 1.13111 Le" 1 . 7 5 1 2 1 1 . 0 0 1 1 1 TVr

2.10121

2 .92131 0 .71111

0 .94111

2 .16121 2 .31121 1 .11111

0 .71111 3.98141

3.34131 2.07121 0.90111

0 .97111 4.00141 3.25141 1 .11111

Table V I : AmlnO acld composltlons of C N B r peptldes of 1 3 0

He f I l e 1.13111

TVr Le" 1 . 7 5 1 2 1 1 . 0 0 1 1 1

2.10121 0 .94111

Pie

LYS 0 .95111 1.00111 1 .15111 w1s

A r g 2.00121 1 .20111 0 .85111 1 .98121 1 .00111

0 . 9 2 1 7 1 0 . 9 8 1 1 1 1.OOlil 1.01111 4 . 0 9 1 4 1

0 .69111

Peptide C8 C 9 C i O C11 Position+ 71-89 90-115 116-124 125-136

Phe

2.00121

2 .03121 1 .75121

2 . 3 5 1 2 )

2 . 7 0 1 3 )

2 . 6 4 1 4 ) 0 .87111

0 . 9 3 1 1 1 0 .88111 2 . 1 4 1 2 ) 2 . 3 5 1 2 1 7 . 4 5 1 6 1

2 .19121 5 .40151

3 . 6 3 1 3 1 2.55121

0 . 8 5 1 1 )

0 .82111 0 . 8 4 1 l l

1 .00111 1 . 0 7 1 1 1

1 . 7 9 1 2 ) 1 . 9 5 1 2 1

0 .84111 1 .82121

2 .06131

1 . 0 0 1 1 ~ 1 . 1 6 1 1 )

Hse 1 1 1 1 . 8 4 1 2 )

H L S 1 . 5 5 1 2 1 0 . 7 5 1 1 ) LY 5 2.67131 A r g HSe * I l l fill

2.47131 4.45151 0.76111

HLS 1.97121 Phe 1 .00111

LY * Arv

1 . 2 9 1 1 ) 0 .74111

1.00111

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THE JOCJRNAL OF B I O L O G I C A L CHEMISTRY :C 1984 hy The American Society of Biological Chemists, Inc. Vol. 259, No. 2, Issue of January 25, pp. 1056-1063, 1984

Printed in U.S.A.

Biosynthesis of Heparin SUBSTRATE SPECIFICITY OF HEPAROSAN N-SULFATE D-GLUCURONOSYL 5-EPIMERASE*

(Received for publication, December 20,1982)

Ingvar Jacobsson and Ulf LindahlS From the Department of Medical Chemistry, Swedish Uniuersity of Agricultural Sciences, The Biomedical Center, Box 575, S-75123 Uppsala, Sweden

John W. Jensen and Lennart Roden From the Institute of Dental Research and Diabetes Research and Training Center, University of Alabama in Birmingham, Birmingham, Alabama 35294

Harry Prihar and David S. Feingold From the Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsyluania 15261

The substrate specificity of heparosan N-sulfate D- glucuronosyl 5-epimerase from a mouse mastocytoma was examined to determine the effects of N-acetyl and 0-sulfate groups on substrate recognition by the en- zyme. [5-3H]Glucuronosyl-labeled heparosan N-sulfate was prepared enzymatically and was modified chemi- cally by partial N-desulfation and N-acetylation. After enzymatic release of tritium, the location of remaining label was determined by deaminative cleavage and analysis of resulting di-, tetra-, and higher oligosac- charides. This analysis indicated that a D-glucuronosyl residue is recognized as a substrate if it is linked at C- 1 to an N-acetylated glucosamine residue and at C-4 to an N-sulfated unit. However, the reverse structure, in which the D-glucuronosyl moiety is bound at C-1 to an N-sulfated residue and at C-4 to N-acetylated gluco- samine, is not a substrate. Similar studies with 0- sulfated heparin intermediates showed that 0-sulfate groups either at C-2 of the L-iduronosyl moieties or at C-6 of vicinal D-glucosaminyl moieties prevent 5-epi- merization. These findings were confirmed by studies of the reverse reaction, in which tritium was incorpo- rated from 3Hz0 into partially 0-desulfated heparin and the location of incorporated radioactivity was de- termined. These and more direct experiments corrob- orated the previous conclusion that the L-iduronosyl moieties are formed after N-sulfation but before 0- sulfation. Assessment of the influence of substrate size on the reaction further showed that a large substrate is preferred; an octasaccharide released tritium at a rate approximately 10% of that observed for the parent polysaccharide, and some release occurred also with smaller oligosaccharides.

The biosynthesis of heparin is a multistep process which includes formation of the nonsulfated polysaccharide, N-ace-

* This work was supported by Grant 2309 from the Swedish Med- ical Research Council, the Swedish Universky of Agricultural Sci- ences, KabiVitrum AB, Stockholm, and National Institutes of Health Grants AM 07069, AM 18160, AM 31101, DE 02670, and HL 11310. This is Paper XI1 of a series in which the preceding reports are Refs. 1 and 2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be sent.

tylheparosan (composed of alternating D-glucuronic acid and N-acetyl-D-glucosamine residues), and several subsequent modifications of this polymeric intermediate. These modifi- cations are initiated by removal of most of the N-acetyl groups and replacement with N-sulfate groups; a large proportion of the D-glucuronic acid residues in the N-sulfated polysacchar- ide is then epimerized to L-iduronic acid, and, finally, 0- sulfation in three positions yields biologically active heparin (3-5).

Of the enzymes involved in the modification process, the uronosyl 5-epimerase has been studied in most detail so far. This enzyme has been purified extensively from a mouse mastocytoma (6) and from bovine liver (7), and some of its molecular and catalytic properties have been determined. An important feature of the reaction mechanism is the exchange of the hydrogen at C-5 of the D-glucuronosyl residues for protons from the medium, as has been demonstrated by the following observations: ( a ) tritium is released from an enzy- matically synthesized heparin precursor polysaccharide in which the C-5 hydrogen atoms of the D-glUCUrOnOSyl residues are specifically labeled with tritium (8, 9), and (b) tritium is incorporated into both D-glucuronic and L-iduronic acid res- idues when polysaccharides containing the appropriate sub- strate structures are incubated with epimerase in the presence of ‘H20 (10, 11).

In a previous study (9) it was shown that N-sulfate is an essential component of the substrate structure recognized by heparosan N-sulfate D-glucuronosyl 5-epimerase and that a glucuronic acid residue may be epimerized if it is located between two N-sulfated glucosamine residues. In contrast, the first polysaccharide product formed during heparin biosyn- thesis (N-acetylheparosan or PS-NAc; see Ref. 9), which contains exclusively N-acetylated glucosamine residues, is not a substrate (9). However, it has not previously been deter- mined whether a particular glucuronosyl residue can be epi- merized if only one of the adjacent glucosamine units is N- sulfated and the other is N-acetylated. Besides the N-acetyl and N-sulfate groups, the 0-sulfate groups may also influence substrate recognition by the epimerase, and circumstantial evidence for an inhibitory effect was obtained in our previous study (9); however, the effect of such groups has not been precisely defined. The present report describes a more detailed investigation of these aspects of the substrate specificity of

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Substrate Specificity of D-Glucuronosyl 5-Epimerase 1057

the epimerase as well as a study of the effect of the molecular size of the substrate.

MATERIALS AND METHODS’~~

RESULTS AND DISCUSSION

Effect of N-Acetyl Groups on Substrate Recognition; Experiment 1

In assessing the role of N-acetyl groups in substrate recog- nition by the epimerase, two different structures should be considered, i.e. 1) -GlcNAc-GlcUA-GlcNSOT- and 2) -GlcNSO:-GlcUA-GlcNAc-. The glucuronic acid residue in the first of these structures is represented by residues 3 and 5 in Fig. 1, while that in the second structure corresponds to residue 4 . To determine whether the two structures are sus- ceptible to epimerase action, an experiment, was carried out which is outlined in Fig. 1 and is described in detail in Miniprint (“Experiment 1”). Briefly, a substrate of high N- acetyl content was prepared from [5-3H,14C]gl~~~r~n~~yl-la- beled heparosan N-sulfate by partial N-desulfation and N- acetylation. After exhaustive incubation with the epimerase, the polysaccharide product was reisolated and treated with nitrous acid, and the resultant di-, tetra-, and higher oligosac- charides were isolated by gel chromatography. In the tetra- saccharide fraction, the nonreducing terminal uronic acid is derived from residue 4 in the substrate, while the internal uronic acid represents residue 5 (see Fig. 1). Following diges- tion of the tetrasaccharide fraction with partially purified p- D-glucuronidase (which also contains a-L-iduronidase), the reaction products (free uronic acid and trisaccharide) were separated by electrophoresis, and their ’H/”C ratios were determined to assess the loss of tritium. As shown in Fig. 1, 29% of the tritium had been lost from the free uronic acid fraction (type 4 residues), but no significant change had occurred in the trisaccharide fraction (type 5 residues). Pro- jected on the intact polysaccharide, this result implies that the sequence -GlcNSO;-GlcUA-GlcNAc- is a substrate for the epimerase, whereas the reverse structure, -GlcNAc- GlcUA-GlcNS0:-, is not. This conclusion is in accord with established structural features of heparin. The sequence -GlcNSO:-IdUA-GlcNAc-. has thus been conclusively iden- tified in both heparin and heparan sulfate (20,22-26), whereas the reverse structure has not been found (however, see Refs. 24 and 26).

The effects of the epimerase on the N-sulfated and the N- acetylated blocks of the substrate were in agreement with previous results (9). The N-sulfated regions (type 6 and 7 residues) lost a substantial proportion of their tritium, whereas the N-acetylated regions (type I and 2 residues) retained the label (Fig. 1; see further discussion in Miniprint).

Portions of this paper (including “Materials and Methods,” part of “Results and Discussion,” Table I, and Figs. 2, 3, 5, 7, 9, and 10) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are readily available from the Journal of Biological Chemistry, 9560 Rockville Pike, Bethesda, MD 20814. Request Document No. 82” 3409, cite the authors, and include a check or money order for $7.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

‘The abbreviations used are: GlcUA, D-glucuronic acid UA, un- specified uronic acid IdUA, L-iduronic acid; GlcNSOT, 2-deoxy-2- sulfamido-D-glucose; aMan, 2,5-anhydro-~-mannitol; PAPS, 3“phos- phoadenylylsulfate; HEPES, 4- (2-hydroxyethyl)-l -piperazineeth- anesulfonic acid. The location of 0-sulfate groups is indicated in parentheses. See Fig. 1 in Ref. 9 for abbreviations of polysaccharides that are intermediates in heparin biosynthesis.

L__

< 5 n d 29 ( 5 68 -

FIG. 1. Schematic outline of experiment designed to deter- mine the effect of N-acetyl groups on the 5-epimerization of adjacent glucuronic acid units. -0-, unspecified [14Clhexuronic slid residue (D-glucuronic or L-iduronic) without 3H label; -0-, [5- 3H,14C]hexuronic acid; -8-, D-ghcosamine residue carrying an acet- ylated (-NAc) or sulfated (-NSO:) amino group; *, 2,5-anhydro-~- mannitol. The potential reducing terminal is to the right. The scheme illustrates the selective loss of 5-3H label from uronic acid residues, as predicted from earlier studies on the substrate specificity of the glucuronosyl 5-epimerase, and also indicates the uronic acid residues (-a-) of previously unknown reactivity that were specifically exam- ined in this experiment. The results given at the bottom are expressed as per cent loss of 3H from the indicated uronic acid residues and wer@‘calculated from the 3H/14C ratios of the degradation products (see Fig. 3 and the text). n.d., not determined. For further information see the text.

Effect of 0-Sulfate Groups Experiment 2A”Does the presence of 0-sulfate on one or

both of the glucosamine units adjacent to a glucuronic acid residue affect the reaction of the epimerase with that residue? An experiment to answer this question is illustrated in Fig. 4. The design of this experiment was similar to that described for the examination of the effect of N-acetyl groups. A radio- active polysaccharide, containing 14C- and 5-3H-labeled glu- curonic acid residues and 6-sulfate groups on some but not all glucosamine residues, was exposed to the epimerase, and disaccharides with or without these 0-sulfate groups were then isolated by deaminative cleavage and analyzed for their content of 3H and 14C. Details of this experiment are described in Miniprint. As shown in Fig. 4 (see residues 1 and 2), the presence of a 6-0-sulfate group on the glucosamine linked to C-1 of a glucuronic acid residue prevented tritium release, i.e. 5-epimerization could not take place. There is some evidence to suggest that this inhibitory effect extends in both direc- tions, i.e. that a glucuronic acid residue is inaccessible to the epimerase even in the structure -GlcNSO:(6-OSO?)-GlcUA- (residue 3). Thus, despite release of all accessible tritium from the substrate polysaccharide during the incubation, about one-third of the nonsulfated glucuronic acid-containing disac- charides in the product were still tritium-labeled. The most reasonable explanation for this finding is the presence of an

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1058 Substrate Specificity of D-Glucuronosyl5-Epimerase

1 2 3 4

NSOS NSO3 NSOg NSO; NS05

3 w j

GhJCUC%OSyl 5 - epinerase

NSOZ NSOS NSOS NS03 NS03

1 HNO2 - NaBH4

os05 os05 +&&"

I I I I

Loss of 3 H (%) I I I I I i - -

<5 67

FIG. 4. Schematic outline of experiment designed to deter- mine the effect of glucosamine 6-0-sulfate groups on the 5- epimerization of adjacent glucuronic acid units. Symbols are as in Fig. 1; in addition, 6-0-sulfate groups are included on some of the glucosamine and anhydromannitol units, as indicated. In this scheme (and in subsequent similar schemes), any effect on the epimerization reaction of 0-sulfate groups more remote from a potential target uronic acid residue than the neighboring glucosamine units has not been considered. The results given at the bottom are expressed as per cent loss of 'H from the indicated uronic acid residues.

inhibitory 6-0-sulfate group on the glucosamine linked to C- 4 of the glucuronic acid residues in question.

Experiment 2B"The inhibitory effect of 0-sulfate groups was verified in another set of experiments (Fig. 6), where the incorporation of tritium from 3H20 into a series of partially desulfated heparin preparations was measured. Upon incu- bation with microsomal epimerase in the presence of 3Hz0, all of the modified heparin preparations incorporated tritium (Table I). The extent of labeling was inversely related to their sulfate contents and decreased from 4730 cpmlpg of uronic acid for an almost fully 0-desulfated preparation to 5-600 cpm for the preparation of highest 0-sulfate content. Char- acterization of the reaction products (see Miniprint) showed that more than 95% of the tritium had been incorporated into disaccharide units which were N-sulfated but not 0-sulfated (Fig. 6). O-Sulfated sequences that were found to be resistant to epimerase action included -G~cNSO,-G~CUA-G~CNSOT(~- OSOS)-, -G~cNS~~-I~UA-G~~NSO~(~-OSO~)-, and -Glc- N S O ~ - I ~ U A ( ~ - O S O . ~ ) - G ~ C N S O T - .

Experiment 2C"An obvious consequence of the experi- ments described above is that the formation of iduronic acid units must precede the incorporation of 0-sulfate groups. This was demonstrated directly by an experiment which consisted of two stages outlined in Fig. 8: ( a ) epimerase-catalyzed in- corporation of tritium from 3H20 into N-deacetylated, N- sulfated heparan sulfate and concomitant O-sulfation by in-

I I 3H incorporated (% of total)

I - <5 -

>95

FIG. 6. Schematic outline of experiment designed to deter- mine the effect of 0-sulfate groups on the uronosyl C5-epi- merization reaction. Symbols are as in Fig. 4; in addition, a 2-0- sulfate group is shown on one of the uronic acid residues (unit I ) . The results given at the bottom show the distribution of 3H label incorporated into various disaccharides units. Units 3 and 4 could not be discriminated by the protocol employed; however, it was tentatively assumed that all label went into the latter unit (see the text).

clusion of PAPS in the reaction mixture; and ( b ) release of accessible tritium by repeated incubation of the products with the epimerase (in the absence of PAPS). Identification of labeled disaccharide units showed that a fraction (13%) of the radioactivity incorporated in the first stage was present in mono-0-sulfated disaccharide units and that no release oc- curred from these units in the second step. In contrast, a control sample without added PAPS did not contain tritium in the mono-0-sulfated units, in accord with the results of Experiment 2B. Given the inherent reversibility of the epi- merase reaction, these findings clearly indicated that incor- poration of tritium had occurred prior to 0-sulfation and that the 0-sulfate groups subsequently attached to iduronic acid and glucosamine units rendered the pertinent uronosyl resi- dues inaccessible to the epimerase.

I t should also be noted that an appreciable fraction of the non-0-sulfated disaccharide units retained their tritium label at the end of the second incubation, supporting the notion that a 6-@sulfate group inhibits epimerization of both adja- cent uronic acid residues (cf. Experiment 2A) (detailed pro- cedures and results of this experiment are described in Mini- print).

Molecular Size of 5-Epimerase Substrate

The influence of substrate size in the epimerase reaction was examined by measuring the release of tritium from oli- gosaccharides containing 5-'HH-labeled uronosyl groups. One group of such oligosaccharides was prepared by digestion of

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Substrate Specificity of D-Glucuronosyl5-Epimerase 1059

1 2 3 4 ;-

NSO? NSOS NSOS NSOB NSOS

3 ~ 2 0 j PAPS o - Wfotransferases

Glucuonosyl 5 - epmerase

oso, ..... .... ..::. Y + Y “ - F

,,,oj GlucumSyl 5 - epimerase

NS0.j 0SO.j NSOj NSOi NSOg NS%

os05

NSOg 0SO.j NS0-j NSOg NS03 NSOS

1 HNO2- NaBH4

os05 +pQ@”+p”

osog

I I I I

- - ? +

FIG. 8. Schematic outline of experiment designed to estab- lish the relation between the 0-sulfation and uronosyl C5- epimerization reactions. Symbols are as in Fig. 6. During the initial phase of the experiment, 0-sulfation and epimerization (tritium in- corporation) took place simultaneously; in the second incubation, the loss of tritium from the previously labeled uronic acid residues was studied. For additional information see the legend to Fig. 9.

[5-:’H]PS-NSO: with bacterial heparitinase (see “Methods”) and chromatography on Sephadex G-50 (data not shown). A second group of oligosaccharides was prepared by acid hy- drolysis of 0-desulfated heparin which had been labeled by incubation with epimerase in the presence of 3H20 (10, 11); after N-sulfation, the resultant oligosaccharides were frac- tionated by chromatography on Sephadex G-25 (see “Meth- ods”).

When tested as epimerase substrates, oligosaccharides of the two series gave essentially the same results. No oligosac- charide was as good a substrate as the intact polysaccharides, and a gradual decrease in tritium release was observed with decreasing size, e.g. a fraction from the heparitinase digest, which was composed essentially of octasaccharide and smaller fragments, released “Hz0 at a rate approximately 10% of that observed for the parent polysaccharide. The tetrasaccharides of either group did not release detectable amounts of tritium, but some release was observed with the penta-, hexa-, and heptasaccharide fractions from the acid hydrolysate, although the measured values were close to the limit of detection. Therefore, we can only conclude safely that a large substrate is preferred and that there is no sharp cutoff point between substrates and nonsubstrates; rather, a gradual decline in substrate activity is observed with decreasing size.

General Discussion

The accumulated knowledge concerning the substrate spec- ificity of the D-glucuronosyl 5-epimerase has been summa- rized in Fig. 11, which shows the polymer modifications leading to a hypothetical, yet plausible nonasaccharide struc- ture in heparin. During this process, the reactivity of the hexuronosyl residues as substrates for the epimerase changes drastically in response to the structural alterations in their immediate environment. Thus, the D-glucuronic acid residues in the fully N-acetylated intermediate, N-acetylheparosan, are not recognized as substrates, but exchange of the N-acetyl for N-sulfate groups generates a structure which is susceptible to attack by the epimerase (9). The present investigation corroborates these earlier conclusions and allows us to further qualify the requirement for N-sulfate groups. Whereas epi- merization of a D-glucuronic acid residue proceeds most rap- idly if both of the adjacent D-glUCOSamine units are N-sul- fated, only the N-sulfate group on the residue linked to C-4 of the uronic acid is an essential component of the substrate structure. Thus, epimerization may occur, albeit at a lower rate, if the D-glucosamine residue bound at C-1 of the D- glucuronic acid is N-acetylated, but the reverse structure is not a substrate. The slow release of tritium from the partially N-acetylated polysaccharide, including its largely N-sulfated regions, further suggests that the reaction rate is influenced by the substitution pattern of glucosamine residues more remote from the uronic acid residues undergoing epimeriza- tion. The likelihood that this is true was strengthened by the observation that a trisaccharide of the appropriate structure was not a substrate and a substantially larger structure was required for maximal reactivity.

Besides the modulation by N-acetyl groups of the reactivity of the hexuronosyl moieties, the effects of 0-sulfate groups were also assessed in the present work. The results clearly

I 2 3 4 5 6 7 0 9

N K Q N P C Q ““0 I I N-deocetylotlon; N-sulfatlon

Glucuronosyl CS-ep~mer~zot~on 2-O-sulfatlon

HkSOi 0’ hK 6 HNSOS “W @SO; HNSO;

0 HNSoi

FIG. 11. Sequence of polymer modification reactions in- volved in the biosynthesis of a hypothetical nonasaceharide sequence in a heparin molecule. 3-0-Sulfated glucosamine units3 have not been included in the structure. The substrate specificity of the glucuronosyl 5-epimerase is illustrated by indicating uronic acid residues that are susceptible to attack by the enzyme (+) and those that are not (-1. Note the glucuronosyl unit 8 that remains unmodi- fied in spite of being a potential substrate for the enzyme.

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1060 Substrate Specificity of D-Glucuronosyl5-Epimerase

showed that a 2-0-sulfated iduronic acid residue is not a substrate for the epimerase and, similarly, that 6-0-sulfation of a D-glucosamine unit abolishes or greatly reduces the reactivity of the uronic acid residue which is attached via the C-1 position of the latter to c-4 of the D-glucosamine unit. Circumstantial evidence further indicated that the inhibitory effect of the 6-0-sulfate group extends to the uronic acid residue on the other side. These features of the substrate specificity correlate well with earlier conclusions (1, 9, 23, 27) regarding the order of the modification reactions and dem- onstrate conclusively that the L-iduronic acid residues must be formed prior to 6-0-sulfation of adjacent D-glucosamine units.3 Additional support for this conclusion came from the experiment illustrated in Fig. 8, which showed directly that uronosyl residues which were initially susceptible to epimer- ase action could no longer react after 0-sulfation of the newly formed L-iduronic acid residues and neighboring D-glUCOSa- mine units had taken place.

The structure of the polysaccharide substrate is not the only determining factor in the selection of target units by the epimerase, and previous studies (27) have indicated that the polymer modification reactions operate at a high level of organization in the cellular membranes, as evidenced by the kinetics of this process. Given a pool of preformed, nonsul- fated, endogenous heparin precursor polysaccharide, the sul- fation and epimerization reactions (initiated by the addition of PAPS) thus do not simultaneously involve all the substrate molecules available; rather, a limited fraction of the pool is rapidly modified at one time. Furthermore, polymer modifi- cation takes place in a stepwise manner, such that certain reactions will be completed within the individual polysac- charide molecule before subsequent reactions are initiated (1, 27). In this process, the association between the C5-epimerase and its polysaccharide substrate appears to be transient; al- though the epimerization reaction is freely reversible (9-ll), it is not allowed to approach equilibrium (9). Instead, only a fraction of the potentially susceptible D-glucuronic acid resi- dues is attacked by the epimerase, each attack leading to the formation of an L-iduronic acid unit which then remains stable. Likewise, once the spared glucuronic acid residues have escaped epimerization, they remain stable. Nevertheless, when polysaccharide containing such seemingly unreactive units is extracted from the microsomal membranes and is then reintroduced to the microsomal enzyme system as an exogenous substrate, these glucuronic acid residues are readily attacked by the epimerase (as evidenced by the loss of 3H from [5-3H,’4C]PS-N/O-S0;; see also Ref. 9). These obser- vations suggest a strict compartmentalization of the intact biosynthetic system, which allows for precisely timed, tran- sient interactions between the various enzymes and polysac- charide intermediates. More information regarding the struc- tural and mechanistic basis for this compartmentalization is clearly required to better understand the biosynthetic process and its regulation.

The antithrombin-binding region in heparin molecules contains a specific 3-0-sulfated glucosamine residue (20,29). While the incor- poration of 3-0-sulfate groups into polysaccharide has been demon- strated in the mastocytoma microsomal system (J. Riesenfeld and U. Lindahl, unpublished results), the temporal relationship between this reaction and the other 0-sulfation reactions has not been established.

Finally, it should be mentioned that besides the enzyme studied here, another D-g~ucuronosyl5-epimerase exists which participates in the biosynthesis of dermatan sulfate (30). Despite the formal similarity in regard to substrate and prod- uct, there is no doubt that the two 5-epimerases are distinct entities, and it is an interesting problem for the future to compare the detailed properties of the two enzymes.

1.

2.

3.

4.

5. 6.

7.

8.

9.

10.

11.

12.

13.

14. 15. 16.

17.

18.

19.

20.

21.

22. 23.

24. 25.

26.

27.

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Proc. Natl. Acad. Sci. U. S. A. 77,6551-6555

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INCUBATKW TIME (hours) 40 60 80 IO

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10 20 30

10 HIORATION DISTANCE ICmJ

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M Kimuraof proteins S5 and L30.

Proteins of the Bacillus stearothermophilus ribosome. The amino acid sequences

1984, 259:1051-1055.J. Biol. Chem. 

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