Proc. Nati. Acad. Sci. USAVol. 91, pp. 1624-1627, March 1994Biochemistry

Dissecting the catalytic mechanism of protein-tyrosine phosphatasesZHONG-YIN ZHANG, YUAN WANG, AND JACK E. DIXON*Department of Biological Chemistry and the Walther Cancer Institute, Medical School, The University of Michigan, Ann Arbor, MI 48109-0606

Communicated by Thomas C. Bruice, November 4, 1993 (received for review August 11, 1993)

ABSTRACT Protein-tyrosine phosphatses (PPases) con-tail an evolutionarily conserved segment of 250 amino acidsrefered to as the P1Pase catalytic din. The recombinantPTPase domainfom Yersuia enterocolitica enhances the rate ofhydrolysis ofp-nitrophenyl phMphate, a ph ate moeser,by approximately 1011 over the non-enzyme-catalyzed rate bywater. Specifi amino acid residues responsible for the catalyticrate latn have been e ned by stedirected mesis. Our results suggest that Asp-356 (D356) and Glu-290(E290)are the general acid and the general base catalysts responsiblefor Yersinia PTPase-catalyzed phosphate ester hydrolysis. TheMPse with both E2P0Q and D356N mutations shows no pH

dependence for catalysi but displays a rate enhan ent of 2.6x 010, compared to the ncatldhydrolysis ofp-nhtrophenylphosphate by water. This rate ehancement probably occurs vMatransition-state stabilizan. Our results suggest that all PIP-ases use a common that depends upoformationn ofa thio ph ate ie dte and general acid-general bas-aayis.

Since the purification and amino acid sequence analysis ofthe first protein-tyrosine phosphatase (PTPase) (1, 2), morethan 30 cDNAs encoding distinct PTPases have been clonedand characterized (3-6). This family of enzymes is involvedin cell-cycle control, cell-cell communication, bacterialpathogenicity, and metabolic regulation (3-6). The PTPasesalso play critical roles in Drosophila development and in thelife cycle of Dictyostelium (7, 8).Amino acid sequence aliment of PTPases suggested that

they contain a common evolutionarily conserved segment ofapproximately 250 amino acids called the PTPase catalyticdomain (3-6, 9). Within this PTPase domain is a signaturemotif, (I/V)VHCXAGXGR(S/T)G (where X can be anyamino acid), that is invariant among all PTPases. Mutationaland chemical modification experiments indicate that theinvariant Cys residue in this PTPase signature motif isessential for enzyme activity (9-11). The Cys residue isdirectly involved in formation of a covalent phosphoenzymeintermediate (12-15). These results suggest that all PTPasesare likely employing a common catalytic mechanism.The Yersinia PTPase (yop5l) was identified in the genus of

bacteria responsible for the plague or the Black Death (9).The yopSl gene and its encoded phosphatase activity areessential for pathogenesis (16, 17). The Yersinia PTPaseshows sequence identity to the mammalian PTPases that ishighest within the catalytic domain (9). We have expressed,purified the Yersinia PTPase in high yields, and obtaineddiffraction-quality crystals of the protein (18). We have alsoundertaken a detailed kinetic analysis of the Yersinia PTPaseto understand the mechanism of phosphate monoester hy-drolyses by this family ofenzymes (48). Yersinia PTPase andmammalian PTPases utilize phosphotyrosine-containing pro-teins/peptides as substrates (1, 2, 9, 19). These enzymes also

hydrolyze the artificial substrate, p-nitrophenyl phosphate(pNPP). The pH-km profile for the Yersinia PTPase cata-lyzed hydrolysis of pNPP is bell-shaped and exhibits twoapparent pKa values of 4.63 and 5.20, respectively. Here weexamine the kinetics of both the wild type and severalsite-directed mutants of Yersinia PTPase: Our results identifytwo acidic residues [Asp-356 (D356) and Glu-290 (E290)J thatappear to participate in general acid-base catalysis. Thesetwo residues are invariant in >30 PTPases amino acid se-quences examined, suggesting that general acid-base catal-ysis is a common mechanism employed by all PTPases.

MATERIALS AND METHODSSite-Directed Mutagenesis. The oligonucleotide-directed

mutagenesis procedure of Eckstein (20, 21) was employedusing an Amersham in vitro mutagenesis system (RPN.1523)to create the desired point mutations. The oligonucleotideprimers used were as follows: E224Q, 5'-GCCTGCGGCG-GTCAAAAGCTAAACCGA; D243N, 5'-GTACGCGC-CAAICTTAATGCC; E276Q, 5'-ATGCTGGCACMAAC-CGAA; E290Q, 5'-TCCAGTTCTCAGATAGCCA; D356N,5'-AATTGGCCCAAICAGACCG; E363Q, 5'-GTCAGCTCTCAAGTTACCAAG; E459Q, 5'-TAAGTTGGCTCAAG-GACAAGG. Bases encoding a mutated amino acid areunderlined. To create the double mutant E290Q/D356N, theKpn I and Nde I fiagment (containing the mutation E290Q)from the pYopSl/pT7/E290Q mutant was excised and usedto replace the identical unaltered fragment from the pYopSl/pT7/D356N mutant. All mutations were verified by dideoxy-nucleotide sequencing (22).

Kinetic Analysis. All enzyme assays were performed at30'C. Buffers used were as follow: pH 3.4-3.8, 100 mMformate; pH 4.0-5.7, 100 mM acetate; pH 5.8-6.5, 50 mMsuccinate; pH 6.6-7.3, 50mM 3,3-dimethylglutarate; pH 7.5-8.7, 100mM glycinamide. All of the buffer systems contained1 mM EDTA and the ionic strength ofthe solution was kept at0.15 M by using NaCl. Initial rate measurements for theenzyme-catalyzed hydrolysis of aryl phosphate monoesterswere conducted as described (18). The Michaelis-Mentenkinetic parameters were determined from a direct fit of the vvs. [s] data to the Michaelis-Menten equation by using thenonlinear regression program ENZFn-rER (23). pH dependencedata were fitted to the appropriate equations by using anonlinear least squares regression program (24). The equationsused were as follows: for the wild-type enzyme, k~a = k/[+ (H+/KP5') + (KO'/H+)]; for E290Q, kmt = kt/[l +(KaPP/H+)]; for D356N, kca = kaf/[l + (H+/KaPP)].

RESULTS AND DISCUSSIONThe hydrolysis of phosphotyrosine-containing peptides andpNPP by PTPases displays a bell-shaped profile when values

Abbreviations: PTPase, protein-tyrosine phosphatase; pNPP, p-ni-trophenyl phosphate; LAR, leukocyte common antigen-related mol-ecule.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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of kt are plotted vs. pH. There are many potential expla-nations for this kinetic data. Perhaps the simplest is that theenzyme utilizes general acid and general base catalysis. Theapparent pKa values noted in the bell-shaped pH vs. rateprofile are =4.6 and z5.2. These values fall within the rangeof ionizations seen for the side chains ofAsp, Glu, or His. Toexplore the mechanism giving rise to the bell-shaped pH vs.

kcat profile, we examined a sequence alignment of the Yers-inia PTPase with several mammalian and Drosophila PT-Pases for invariant Asp, Glu, or His residues (9). Only oneHis residue and seven acidic residues (Asp and Glu residues)were invariant in the PTPase catalytic domain (9). Theinvariant His residue in PTPases is adjacent to the catalyti-cally essential Cys residue. This His residue has been alteredby site-directed mutagenesis (10-12, 25, and Z.-Y.Z. andJ.E.D., unpublished data). The activities of several ofthe His"mutant" phosphatases were 0.1-10% that of the wild-typeenzyme, depending on the specific amino acid substitution.Importantly, replacement of the His residue by either Asn orAla did not alter the characteristics ofthepH vs. rate profiles,although the pH optima were shifted due to perturbations ofPKa values of catalytic groups (Z.-Y.Z. and J.E.D., unpub-lished data). Based on these analyses, it seems clear that theinvariant His residue is not a general acid-base catalyst. Inthis report, we focus our attention on the seven conservedacidic residues in the Yersinia PTPase domain, namely,Glu-224, Asp-243, Glu-276, Glu-290, Asp-356, Glu-363, andGlu-459. We made the most conservative site-directed mu-tations possible (i.e., Glu -- Gln and Asp -* Asn) to remove

the ability of these residues to function effectively in generalacid-base catalysis while minimizing structural perturbationsin the proteins. All of the mutations were verified by DNAsequencing, and the proteins were expressed in Escherichiacoli (18) and purified to homogeneity as judged by SDS/PAGE (data not shown). Table 1 summarizes kinetic param-eters, k., and Km, of the wild-type and the mutant enzymesat pH 5.0, 6.0, and 7.0, by using pNPP as the substrate. It isapparent that mutant PTPases E224Q, D243N, E276Q,E363Q, and E459Q had kcat and Km values effectively iden-tical to those of the wild-type Yersinia PTPase. In contrast,the PTPase mutants E290Q and D356N had kw values thatwere drastically different from those of the wild-type en-zyme. We consider it unlikely that the altered activity of theenzymes E290Q and D356N was a consequence of alteredstructure, since both proteins had chromatographic proper-ties, UV spectra, and Km values for pNPP that were similarto those of the wild-type enzyme.

Fig. 1 shows the pH dependence of k,,.t values for the twomutant PTPases, E290Q and D356N, and the native enzyme.It is evident that the point mutations at residue 290 or 356 alterthe pH vs. kct profiles when compared to the pH vs. kctprofile for the wild-type enzyme. The maximum turnovernumber for the wild-type enzyme was decreased from 2114 +250 s-1 to 17.9 ± 1.5 s-1 and 0.929 ± 0.044 s-1 for the mutantPTPases E290Q and D356N, respectively. Elimination of the

A A0 A Aa

1X0

0.1

3.0

pH

FIG. 1. pH dependence of kc of the Yersinia PTPase (o) and themutants E290Q (A), D356N (O), and E290Q/D356N (a).

carboxyl group at residue 290 (E290Q) caused the disappear-ance of the acidic limb of the pH profile, whereas eliminationof the carboxyl group at residue 356 (D356N) caused thedisappearance of the basic limb of the pH profile. Thecatalytic efficiency of the mutant E290Q is dependent on anionizing group with an apparent pKa of 5.16 ± 0.06, whichmust be protonated for catalysis. This pKa value is almostidentical to the pK21w value (5.20 ± 0.06) of the wild-typeenzyme. On the other hand, the mutant D356N retained anionizing group with an apparent pKa of 4.70 ± 0.04 that mustbe deprotonated for catalysis. This pK4 value is effectivelyidentical to the pKfPP value (4.63 ± 0.07) of the wild-typeenzyme. Thus, mutations at either residue 290 or 356 in theYersinia PTPase led to a marked reduction in catalyticactivity and an alteration in the pH dependence for catalysis.These results suggest that E290 acts as a general base and thatD356 acts as a general acid in the Yersinia PTPase catalyzedreaction. The bell-shaped pH dependence of kt is notrestricted to the Yersinia PTPase. In fact, we observed thatboth the mammalian intracellular PTPase (PTP1) and thereceptor PTPase (LAR, leukocyte common antigen-relatedmolecule) had similar kinetic properties (Z.-Y.Z. and J.E.D.,unpublished data).To further substantiate our proposal that E290 acts as a

general base and D356 acts as a general acid in PTPasecatalysis, we altered both residues within the PTPase domainby site-directed mutagenesis (E290Q and D356N). This re-combinant protein (E290Q/D356N) was obtained in a homo-geneous form as noted above. The E290Q/D356N PTPaseexhibited kct values that were lower than either single mutant(Fig. 1 and Table 1). More importantly, the elimination ofboth carboxyl side chains at residues 290 and 356 renderedthe PTPase-catalyzed phosphomonoester hydrolysis pH-independent from pH 4.6 to pH 8.4 (Fig. 1). This is in accordwith the conclusion that residues E290 and D356 function asthe general base and the general acid, respectively, and these

Table 1. Summary of kinetic parameters of the Yersinia PTPase and its mutants of the conserved acidic residues

Yersinia pH 5.0 pH 6.0 pH 7.0

PTPase kcat, s- Km, mM kcats5- Km, mM kcat, S-1 Km, mMWild type 1230 ± 29 2.55 ± 0.17 345 ± 5.5 2.60 ± 0.12 34.2 ± 2.6 2.90 ± 0.52E276Q 1664 ± 89 3.98 ± 0.49 415 ± 11 3.82 ± 0.24 56.4 ± 2.0 2.40 ± 0.23E290Q 18.7 ± 1.1 6.70 + 0.71 1.90 ± 0.06 4.43 ± 0.33 0.311 ± 0.01 3.79 ± 0.24D356N 0.720 ± 0.02 3.65 ± 0.23 0.891 ± 0.02 3.38 ± 0.20 0.894 ± 0.04 3.28 + 0.30E224Q 1044 ± 34 1.70 ± 0.17 343 ± 11 2.59 ± 0.22 39.2 ± 4.3 2.62 ± 0.07D243N 1098 ± 1.5 2.37 ± 0.08 313 ± 5.2 1.99 ± 0.10 38.8 ± 0.10 2.89 ± 0.02E363Q 1242 ± 30 2.36 ± 0.15 342 ± 7.1 2.18 ± 0.13 37.9 ± 0.07 2.97 ± 0.01E459Q 1173 ± 15 2.38 ± 0.10 315 ± 2.7 2.14 ± 0.05 36.0 ± 3.9 3.13 ± 0.78E290Q/D356N 0.0603 ± 0.0031 5.7 ± 0.72 0.0468 ± 0.0012 3.88 ± 0.24 0.0409 ± 0.0008 2.55 ± 0.14

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two residues are responsible for the apparent pKa valuesnoted for the bell-shaped pH rate profile seen for the familyof catalysts. The E290Q/D356N PTPase had Km values forpNPP that were effectively indistinguishable from the wild-type enzyme (Table 1), suggesting that there are no majorstructural alterations in the E290Q/D356N PTPase.Table 2 shows the relative maximum turnover number of

Yersinia PTPase and the mutants E290Q, D356N, andE290Q/D356N in comparison with the uncatalyzed waterhydrolytic rate seen with pNPP. With the rate of waterhydrolysis ofpNPP set at 1.0, Yersinia PTPase accelerate theuncatalyzed pNPP hydrolysis (26) by a remarkable 1.4 x1011-fold. Replacement of E290 with Q caused a reduction of130-fold in kct and replacement of D356 with N caused areduction of 2.4 x 103-fold in kz t. When both E290 and D356were replaced with Q and N, respectively, the kct value wasreduced by 5.4 x 104-fold. Interestingly, the E290Q/D356NPTPase retains a catalytic activity that is 2.6 x 106-fold abovethe uncatalyzed hydrolysis rate seen with water (Table 2). Wepropose that this residual activity results from transition-state stabilization. Similar transition-state stabilization ef-fects have been observed with the serine protease subtilisinwhen the catalytic triad was completely abolished (27). Theturnover number of the double mutant is in the range ofthosefound for "good" catalytic antibodies (28-30). The E290Q/D356N PTPase reaction probably proceeds through a phos-phoenzyme intermediate that is then slowly broken down bywater. Proof of this awaits additional experimentation.Our kinetic data suggest that D356 and E290 in the Yersinia

PTPase function as a general acid and a general base,respectively. We realize that proof of this suggestion willrequire data in addition to the pH vs. kcat results reportedhere. To gain further insights into the possibility that allPTPases might use acidic residues equivalent to D356 andE290 in catalysis, we reexamined the amino acid sequencealignment of the PTPases. Earlier alignments of the PTPaseswere either confined by the availability of known proteinsequences or based on sequences from closely related speciesthat overemphasized sequence similarity (9, 31, 32). A se-quence alignment of bacterial, yeast, and mammalian PT-Pases was undertaken to determine whether the suggestedcatalytic properties of the two acidic residues essential forgeneral acid-general base catalysis of the Yersinia PTPaseswere invariant among all PTPases reported to date. Thisalignment provides considerable insight into residues thatmay be essential for catalysis or structural integrity ofPTPases. As shown in Fig. 2, there are 21 residues that areinvariant among all PTPases. Ofthe seven "invariant" acidicresidues identified from the earlier alignment (9), only two areinvariant in alignments of all known PTPases (i.e., E290 andD356). This is consistent with the experimental results de-scribed in this report that indicate that E290 and D356 havedramatic effects on catalysis. This suggests that all PTPasesare likely to employ general acid-general base catalysis,utilizing residues at positions equivalent to those of E290 andD356 in the Yersinia PTPase.We would like to point out that the PTPase sequence

alignment shown in Fig. 2 does not include the dual-specificity phosphatases, which are capable of removing

Table 2. Change in kat of the Yersinia PTPase mutants usingpNPP as a substrate

Yersinia PTP Relative maximal turnover numberWild type 1.4 x 1011E290Q 1.1 x 109D356N 5.9 x 107E290Q/D356N 2.6 x 106H20 1.0

23.. .. S

.- s-. SS

... snm...... .... S

Cr p

.Y. TIC P

:Y T A; PP

- TCi

P

T P

. , .

.vJI R TA.

I IV

*L'V IiI I 1 S7iE STG : : .. -

;m-3ivLV' VSl 'dG --

IV.Vl.. SV, T .: :

L I &.Lv T'v:L

..7:.L .EMI 3 F1 L I a;

VV?4S HVV S-M

FIG. 2. PTPase domain alignment. Sequence alignments of thePTPase domain of Yop5l [Yersinia PTP (33)], YPIP1 [yeast PTP1(34)], YPTP2 [yeast PTP2 (35)], hTcell [human T-cell PTP1 (36)],rPTP1 [rat PTPI (37)], hPTP1C [human PTP1C (38), SH2 domain-containing PTP], rLAR [rat LAR (39), a receptor-like PTP; only thePTP domain 1 was used in the alignment], Megal [humanPTmegal(40), cytoskeleton binding domain-containing PTP], andPEP [PTP ofhematopoietic origin, rich in PEST sequence (41)]. Residues in theblack boxes represent absolutely invariant residues among all ninePTPs from bacteria, yeast, and mammals. Residues in shaded boxesindicate conservative substitutions. Gaps are introduced as dots. Fororientation purposes, starting and ending sites are as follows: Yop5l,residues 219-462; YPTP1, residues 48-329; YPTP, residues 404-738; hTcell PTP, residues 38-276; rPTP1, residues 36-278; hPTP1C,residues 268-518; rLAR, residues 328-563; PTP megal, residues675-912; PEP, residues 50-290.

phosphate from phosphoserine/threonine- and phosphoty-rosine-containing proteins. This subfamily of phosphatasesincludes the originally described dual-specific phosphatasefrom vaccinia virus, known as VH1 (42) and the cell cycleregulatory protein cdc25 (6). Although this subfamily ofphosphatases shows sequence identity to the PTPases in thePTPase signature motif, it generally has limited sequenceidentity to other PTPases outside the active site. Due to thelimited number of VH1-like phosphatases known, we havenot yet identified the corresponding conserved acidic resi-dues for this group of enzymes.The PTPase alignment shown in Fig. 2 also highlghts other

invariant residues within the PTPase catalytic domain. TheNH2-terminal sequence of the PTPase domain contains three

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highly conserved regions, namely, NRYX(N/D)(I/V), DYINA,and YIX(C/T)QXP (boldface type designates invariant resi-dues;X designates any amino acid; and other symbols designateconservative substitutions or positions that were present ineight out of nine alignments used in the comparison shown inFig. 2). Random chemical mutagenesis using hydroxylamineand N-methyl-N'-nitro-N-nitrosoguanidine performed with thefirst cytoplasmic PTPase domain of human LAR (43) hasidentified eight temperature-sensitive mutants clustered in thisregion of the PTPase. The "middle" of the PTPase domaincontains the two conserved acidic residues, E290 and D356, thatappear to function as general base and general acid catalysts,respectively. The COOH-terminal sequence of the PTPasedomain contains two highly conserved regions. The first is thePTPase signature motif, PX(I/V)(I/V)HCSAGXGR(T/S)G,which includes the catalytically essential C403 that is involvedin the nucleophilic attack on the phosphorus atom in thesubstrate; H402, which may serve to stabilize the active sitethiolate anion (44); and the GXGXXG sequence, which mayplay a role in substrate binding based upon its similarity to thenucleotide binding fold seen in dehydrogenases (45) and kinases(46). The second conserved region located in the COOH ter-minus includes two invariant Arg residues, R437 and R440, andtwo invariant Gln residues Q446 and Q450. The two Argresidues, R437 and R440, together with R228 and R409 couldinteract with the negatively charged phosphate present on thesubstrate or the transition state. Alternatively, one or more ofthe Arg residues could play a role in the interaction with thearomatic Tyr residue of the substrate (47) as is seen with SH2domain-containing proteins.Our results have led to the identification of two specific

amino acid residues (E290 and D356) that appear to functionin general acid-general base catalysis ofthe Yersinia PTPase.To the best of our knowledge, this provides the first examplein which site-directed mutagenesis has identified residuesthat appear to be directly involved in general acid-basecatalysis in the absence of any three-dimensional structuralinformation. These two residues are invariant in all PTPasesexamined to date, suggesting that all PTPases use this com-mon mechanism for catalysis.

We thank Dr. Kevin Walton and Dr. David Hakes for their helpduring the PTPase domain amino acid sequence alignment. We alsothank Dr. Randy Stone for his comments on the manuscript. Thiswork was supported by National Institutes of Health Grant 18849,the General Clinical Research Center Grant M01-RR00042, and theWalther Cancer Institute.

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