THE ROLE OF COMPLEX METAL IONS IN THE YEAST HEXOKINASE REACTION*

BY NORTEN C. MELCHIOR AND JACKLYN B. MELCHIOR

(From the Departments of Biochemistry and Pharmacology, Graduate School and the Stritch School of Medicine of Loyola University, Chicago, Illinois)

(Received for publication, May 8, 1957)

It is known that many of the enzyme systems which ut,ilize ATPl are activated by inorganic cations. In most instances a divalent cation is required (2) and in some cases monovalent cations are also required (3-5). These effects have usually been assumed to be due to activation of the enzyme by the cation.

The demonstration that many of these cations react with ATP to form complex ions (6-10) makes it necessary to consider these molecular species in any attempt to interpret the activating or inhibiting effects of metal ions on enzyme systems which utilize ATP. Melchior (8) has pointed out t.he variety of molecular configurations which are to be expected in solu- tions of ATP in the presence of various positive ions, emphasizing the possibility that one of these configurations may be preferred or required by the enzyme.

The detailed mechanism of the interaction of magnesium and ATP with various kinases has not been established. Hers (7) suggested that the magnesium-ATP complex was the specific substrate for liver fructokinase, Liebecq (11) reached a similar conclusion in the case of muscle hexokinase, Lardy and Parks (12) pointed out several possibilities in connection with their work on liver fructokinase, muscle phosphohexokinase, and ATP- creatine transphosphorylase, and Raaflaub and Leupin (13) concluded that MgATP-2 is not the substrate for yeast hexokinase. None of these authors presented a quantitative analysis of their dat,a.

* This investigation was supported by a research grant (No. H-1342C3) from the National Heart Institute of the National Institutes of Health, United States Public Health Service. Portions of this material have been presented before the Division of Biological Chemistry at the 123th national meeting of the American Chemical Society and before the American Society of Biological Chemists (1).

1 The following abbreviations will be used in this paper: ATP, adenosine triphos- phate; ATP+, HATPa, NaATP-8, MgATP-2, etc., refer to specific ionic species which exist in certain aqueous solutions containing ATP; TMA+, tetramethylam- monium ion; TMACl, tetramethylammonium chloride; TEA, t.riethanolamine. Pa- rentheses enclosing a molecular formula indicate the concentration of that species in millimoles per liter unless stated otherwise; the sum of the concentrations of all the molecular species containing a particular component is indicated with the sub- script T, e.g. (ATP)T. To simplify the mathematical notation in certain equations, A will be used instead of (ATPO, B instead of (Mg++), and C instead of (MgATP2).

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610 COMPLEX METAL IONS AND YE24ST HEXOKINASE

In order to determine the role of a metal ion in a particular enzyme system it is necessary to know much more than the effect of changes in the total metal salt concentration upon the rate of the catalyzed reaction. The kinetic data must be interpreted in terms of the actual concentrations of the various molecular species present during the measurements. The concentrations of the molecular species present in an aqueous solution (pH 2 7) which contains MgClz and tetraalkylammonium ATP can be

TOTAL MAGNESIUM, MM FIG. 1. Effect of (Mg)r on the concentrations of several of the molecular speciex

in a solution in which (ATP)T is 1 mM, pH 8.6.

determined by solving the following simultaneous equations, most conven- iently by approximations

(I) (ATP)T = (HATPa) + (ATF?) + (MgATP-*)

(II)

(III)

(n@T = (nk*) + @kATP*)

K (H+) x (ATE’-9 aEATTa =

(HATP-3) = 1.26 X lo-’ mai (8)

(IV) KIM~ATP-2 = (MgATP-2)

(Mg++) X (ATP-4) = 2o mN-’

The value for K fMpATP-2 was determined in this laboratory and is in ex- cellent agreement with the value reported by Martell and Schwarzenbach (10) when their results are corrected for the KATP3 complex (8) present in the 0.1 M KC1 which they used as a background electrolyte.

The results of such calculations (Fig. 1) demonstrate the marked differ- ence between (Mg)T and (Mg++) caused by 1 mM (ATP)T, and the strik- ing change in (ATP4) caused by the addition of magnesium. The con-

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N. C. MELCHIOR AND J. B. MELCHIOR 611

centration of HATI’-” (not shown) at a given pH is a constant fraction of (ATP4). Fig. 2 shows the dependence of the concentrations of the various molecular species upon both (ATP)T and (Mg)T.

For a number of reasons it may be desirable to measure enzymic reac- tions in the presence of presumably inert salts. Since Melchior has dem- onstrated (8) that both sodium and potassium ion form complexes with ATPs4, it is necessary to consider what effect the presence of these salts

I

(ATP-4) ,

(MG”)

Lk 0.5 I

2.5

5

0 2 4

I I

(MGATP-~)

5 s 2

I 0.5 r

I I

2 4

TOTAL MAGNESIUM MM FIQ. 3. Effect of (Mg)T on the concentrations of several of the molecular species

present in solutions of ATP at pH 8.6. (ATP)T, 0.5 to 5 mq as indicated on the individual curves.

would have on the hinds and concentrations of the molecular species present in the magnesium-ATP system. For this purpose it is necessary to add the following equations to the system to be solved

(VI (Na)T = (Na+) -I- (NaATYa)

WI) K (NaATP-8)

/N&.4TPm8 = tNa+l x tATp41 = 0.01 m&i-’ (8)

Fig. 3 indicates that moderate concentrations of sodium ion have a signifi- cant effect on the concentrations of the species present in a given mixture of ATP and magnesium and add comparable amounts of a new ATP- containing species. Potassium ion would have a similar effect. This suggests that it might be wise to avoid these cations in investigations of magnesium effects on enzyme systems which utilize ATP.

It was the purpose of this investigation to determine the extent to which quantitative correlation of molecular concentrations with measured

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612 COMPLEX METAL IONS ANI) YEAST HEXOKINASE

react,ion rates could clarify the mechanism of the magnesium activation of t,he yeast hexokinase-catalyzed reaction between glucose and ATP. It was found that t.he initial velocity measured at pH 8.3 to 8.6 over a wide range of (Mg++) and (ATP4) could be quantitatively represented by a simple equation. Several mechanisms were found which yielded kinetic equat,ions of the same form as that derived from experiment. Each of these mechanisms included the formation of a complex involving ATP-4 and the enzyme. Since all these possibilit,ies yield kinetic equations of the same form, measurements of t,his type cannot decide between the

TOTAL SODIUM, M FIG. 3. Effect of t,he addition of sodium chloride on the concentrations of several

of the molecular species in a solution in which (Mg)T = (ATP)T = 1 mM. (Na+) is approximately equal to (Na)T.

mechanisms. None of these mechanisms permits appreciable reaction of hIg++ with the active site of the enzyme without ATP.

Quantitative interpretation of t,he effect of substitution of Na+ for T&IA+ upon the rate of t,he yeast hexokinase-catalyzed reaction indicates that Xa+ probably inhibits by reacting with an enzyme-ATP complex. This is an argument in favor of one of the mechanisms mentioned above, but it is not conclusive.

Consideration of the kinetic equation which describes the experimental results made possible a correct prediction of the change of velocity of the reaction with change in ionic strength at low ionic strengths, and the predict,ion that, fluoride ion must be inhibitory under appropriate condi- tions. This prediction was not in accord with statements appearing in the literature (14), but has been verified experimentally (15).

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N. C. MELCHIOR AND J. 3. MELCHIOR 613

EXPERIMENTAL

The enzyme preparation was Sigma type III yeast hexokinase, specified to contain at least 123,000 units per gm. (16). Spectrophotometric meas- urements were made with a Beckman DTJ spectrophotometer equipped with photomultiplier and thermostatic control of the cell compartment.

The TMA salt of ATP was prepared by shaking BazATP (Pabst) with Dowex 50 W-X-8 cation exchange resin in the TMA form. The concen- tration of the barium-free stock solution was determined from its absorp- tion at 2590 A (slit 0.03 mm). Eastman 1599 TEA was redistilled in vucuo. Eastman 3592 TMACl was recrystalliied twice from 2-propanol, once from 95 per cent ethanol, and dried in vacua. Water was redistilled in a Pyrex glass apparatus; obher chemicals were reagent grade.

The assay system depended upon the fact, first reported by Colowick and Kalckar (17), t,hat 1 mole of hydrogen ions is produced per mole of glucose phosphorylated by ATP in the presence of yeast hexokinase. A convenient assay method for hexokinase activity which involves this prin- ciple has been developed by Colowick and Darrow.2 Final concentrations in the measurements which are reported here were TEA, 2.00 or 4.00 mM; cresol red 4.24 y per ml.; glucose 1.0 or 2.0 mM; final volume 3.00 ml.; temperature 25.0’; pH 8.3 to 8.6. The reaction was started by the addi- tion of the enzyme dissolved in aqueous glucose: and the rate of decrease of the concentration of the salt form of cresol red was measured spectro- photometrically at 5710 A. From the known quantities of indicator and TEA (total) in the system and the apparent pK values of both substances (measured at the ionic strength of the reaction mixture), it was possible to calculate the number of moles of acid produced which corresponded to the measured reduction in absorption at 5710 A. When (ATP)T exceeded (Mg)T, it was necessary to make a small correction for the buffering capac- ity of the ATPm4 present. The initial velocity was determined from the slope of a graph of micromoles of acid produced versus time during the early stages of t,he reaction. Fig. 4 shows typical experimental data. Va is expressed as micromoles per liter per minute for an enzyme concentration of 1 y per ml., and was a linear function of enzyme concentration. The values used for the quantitative kinetic analysis of this system (see Fig. 9 a.nd under “Discussion”) were the averages obtained on different days with different samples of the same enzyme preparation. The maximal standard error of any V. value was less than 3 per cent. In the absence of fluoride ion, the order of addition of reagents did not measurably affect Vo. Chang- ing the glucose concentration from 1 to 2 mM did not affect Vo, which is

1 The authors wish to thank S. I’. Colowick and R. A. Darrow for providing the details of their useful method in advance of publication.

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614 COMPLEX METAL IONS AND YEAST HEXOKINASE

consistent with the Michaelis constant for glucose reported by Slein et al.

(18).

Results

E$ect of ATP-Fig. 5 shows that at constant (Mg)T the initial velocity of the yeast hexokinase-catalyzed reaction increased with (ATP)T until (ATP)T approximated (Mg)T. Further increases in (ATP)T resulted in a decrease in velocity. The maximal rate observed depended upon (Mg)T.

0 IO 20 I I I I I 2

MINUTES MM (ATA,

FIG. 4 FIG. 5

FIG. 4. Reproducibility of the determination of the initial velocity of the yeast hexokinaae-catalyzed reaction between glucose and ATP. 0, (ATP)r = 5.0 mllr, (Mg)r = 10 mM. 0, (ATP)r = 0.50 mM, (Mg)r = 0.10 mM. Acid production is expressed in micromoles per liter for a solution which contains 1 y of enzyme per ml. Each group of experimental points has been displaced along the time axis, the first point in each group was determined exactly 2 minutes after beginning the addition of the enzyme to the reaction mixture. The slopes of the lines through the experi- mental points plotted are, from left to right, 1.55, 1.53, 0.45, 0.43.

FIG. 5. Effect of (ATP)r on the initial velocity of the yeast hexokinase reaction. (Mg)r is indicated on each curve; (TMACI) = 0.3 ar; other conditions are given under “Experimental.” The lines have no theoretical significance.

E$ect of Jfg-Fig. 6 shows the effects of various levels of (Mg)T upon V, at several values of (ATP)T. It should be noted that excess Mg was not inhibitory and that the maximal velocity depended upon (ATP)T.

E$ect of Nu-Fig. 7 shows the effect on Vo of substitution of Na+ for TMA+. The rate of reaction is decreased whether (Mg)T/(ATP)T is 0.5, 1.0, or (not shomrn) 1.5. Similar results were obtained with Kf.

E$ect of Change of Ionic Strength--Fig. 8 shows that relatively large changes in ionic strength have only small effects on V, unless (ATP)T exceeds (hfg) T .

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N. C. MELCHIOR AND J. B. MELCHIOR 615

1.0 4

0 0 >” >f 0

\ 05 . l 0

‘--1

0

2 9 l

0 2 4 0.1 0.2 0.3

MM (MG)T (NA$, MOLAR

FIG. 6 Fxo. 7 FIG. 6. Effect of (Mg)r on the initial velocity of the yeast hexokinase reaction.

(ATP)r is indicated on each curve. Other conditions are as for Fig. 5. FIG. 7. Demonstration of inhibition of the yeast hexokinase reaction by a so-

dium-containing species. 0, lower line, (Mg)r = 0.59 mM; 0, upper line, (Mg)T = 1.0 mnr; (ATP)r = 1.0 mu; NaCl was substituted for TMACl at an ionic strength of 0.3. Other reaction conditions are given under “Experimental.” The circles represent experimental determinations, the lines represent the velocity calculated from Equation 3 according to changes in (MgATP-2) and (ATY’) (see Fig. 3) caused by substitution of Na+ for TMA+.

I

0 5 IO

IONIC STRENGTH I/(MGATP-~) FIG. 8 FIG. 9

FIG. 8. Effect of (TMACl) on the initial velocity of the yeast hexokinase reac- tion. (ATP)r is 1.0 m&r; 0, (Mg)T = 2.0 mu; O,l.O mM; a, 0.50 my. Other condi- tions are standard.

FIG. 9. Experimental basis for Equations 1, 2, and 3. 0, (ATP)r = 0.50 mM; l , (ATP)r = 5.0 mM. The lines were calculated from Equation 3 by using the constants given in footnote 3. Conditions not shown are standard; (TMACl) = 0.3 M.

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616 COMPLEX METAL IONS AND YEAST HEXOKINASE

DISCUSSION

Quantitative RelationshipsMeasurements of the effects on V. of chang- ing (Mg)T at constant (ATP)T and of changing (ATP)T at constant (Mg)T were treated analytically on the basis of several different assumptions concerning the identity of the molecular species which interact with the enzyme in the rate-determining process. It was found that a plot of l/V, versus l/(MgATP+) was linear when (Mg)T was varied at constant (ATP)T (Fig. 9). The slope of the line depended upon (ATP)T, but the intercept on the ordinate axis was not affected by (ATP)T. V0 was also linear with enzyme concentration, but no other simple relationship was found between V. (or l/V,) and any molecular species present.

The data shown in Fig. 9 can be represented by the equation

1 _ = vo

K 1

+ K + Ks(ATI')T~

c

The meaning of this relationship is not clear until the term (ATP)T is translated into concentrations of molecular species which are actually present. At the pH of these measurements the relationship (ATP)T = 1.02A + C accounts for the major ATP species’ present, and substitution of this into Equation 1 gives

1 v, = (Kl + &I +

(Kz ,+ 1.02KaA) c

Although this equation is in the form which is expected (19) if MgATPp2 (C) were the specific substrate and ATP-4 (A) a competitive inhibitor, the possible interactions of the substances present in this system (shown sche- matically in Fig. 10) are too complex to permit such a conclusion unless other pathways to the enzyme-substrate complex can be eliminated.

For convenience in comparing the experimental results with equations based on possible reaction mechanisms, Equation 2 can be written3

(3) vo c -= VllI (C + Q + B-4)

8 The relationships of the constants in Equations 2 and 3 are clearer when Equa- tion 3 is written in reciprocal form:

(3a)

It follows that (Kr + Ka) = l/V,; a/V,,, = Kz; p/V,,, = 1.02K3. According to Equa- tion 3, the values of (r and p should be independent of the specific activity of the en- zyme preparation, and we have verified this with two preparations whose specific activities differed by a factor of 2.5. In both cases 01 = 0.13 and j3 = 0.27 at an ionic strength of 0.3 (VO in micromoles per liter per minute at an enzyme concentration of 1 y per ml., other concentrations being in millimoles per liter).

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N. C. MELCHIOR AND J. B. MELCHIOR 617

The equation for the steady state velocity4 for the system shown in Fig. 9 is in a form similar to Equation 3, but contains terms in A2, B, B2, etc., which cannot be eliminated without assumptions concerning the relative values of the various rate constants which are tantamount to the elimina- tion of certain of the reaction pathways shown. Since Equation 3, the experimental relation between V. and certain concentrations, does not contain such t.erms, we must conclude that not all the pathways shown in Fig. 10 are important in this system. Several different schemes derived by eliminating one or more of the reaction paths yield steady state equa- tions which are compatible with Equation 3 and are, therefore, possible mechanisms for this reaction. These equations follow.

‘(13 ES - E+P

E2 Fro. 10. Diagram of some of the relationships between molecular species present

in an enzyme-catalyzed reaction involving two substances, A and B, which can form a complex, C. In this paper, A refers to (ATP-*), B to (Mg*), C to (MgATP*), and E represents the enzyme or enzyme-glucose complex. The symbols over the arrows include the rate constant,s appropriate for the conversion indicated. For example, in the reaction E + A 8 Et, the rate of formationof El at any instant by this pathway would be given by k&‘A and the rate of the reverse reaction by kaEl.

I. If it is assumed that lC2 is not formed, the two upper pathways to ES remain and the steady state equation is

vo ksk,AB + k3ksC + kpk,BC (4)

-= V, ksk,AB + k&C + k,k,BC + kcsA(k( + ks -6 kn)

+ I&C + ko(kc + ks + kd + k,Bh + kn)

4 The convenient method of King and Altman (20) was used to write the steady state equat.ions for the various systems considered. The complexity of the mathe- matical expressions was reduced by considering only terms involving the reactants and by assuming, in agreement with experiment, that kl is relatively large. This limits the equations to systems which contain no product. We have indicated this by using Vo in the equations, and have measured the initial velocity in the absence of significant amounts of reaction products. It is important to note that the con- siderations of Segal et al. (21) concerning a similar situation are not helpful in re- ducing the complexity of the system treated. Lack of symmetry of the kinetic effects of the components of a complex shows that interaction of the complex with the enzyme is not the only interaction with the enzyme; it does not show interaction of the complex with the enzyme to be absent.

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618 COMPLEX MET.4L IONS AND YE.4ST HEXOKINASE

The terms in B and BC and one term in C do not appear in Equation 3. Therefore this mechanism is not in accord with the experimental findings unless it is further limited. In order to eliminate the incompatible terms, ceptain limits must be placed on the k’s contained in these terms so that, within the concentration range investigated, these terms are negligible wit,h respect to others in the expression. This means that both JG5 and ks must be considerably larger than each of the other k’s. In this way, any term not including either kg or Ice will be negligible and the equation will reduce to

(5) VO ksk,AB + kak& -= V** ksks4B + kc;k& + (ka + kbA)(& + ka + k,z)

Since C = KAB, this equation is of the same form as Equation 3. conse- quently the experimental findings are compatible with the simultaneous operation of the two upper pathways in Fig. 10.

II. If it is further assumed that there is no appreciable conversion of & to ES by reaction with Mg++, i.e. that k7 is negligible, the equation becomes

(f-3 VO kak&’ -= V, L&C + (h + ksA)(k, + k31

This equation is compatible with the experimental results and corresponds to the idea that MgATP2 is the specific substrate and ATP4 is a competi- tive inhibitor.

III. If we assume t,hat h’s is formed only by reaction with MgATF2 but can dissociate Mg++ to form h$, we obtain

(7) VO ksk?BC -= Vlll [ksk,BC + k,B(ka + kn) + ksksC1

At first glance it appears that this equation contains so many terms in B that comparison with Equation 3 is ridiculous, but if one divides numera- t,or and denominator of the right hand fraction by B and uses the relation- ship, C = KAB, to evaluate the term C/B, Equation 7 becomes

(8) VII ka.bC -_ = V, L’&C + kr(h + ha) + k&&U

This equation represents a most unusual molecular mechanism, but it conforms to the experimental findings and cannot be excluded on kinetic grounds.

IV. If one assumes t,hat the uppermost pat,h is the only route to ES, one obtains

(9) VO ksk,AB -= VW2 [k&AB + (ke + ksA)(ks + ha) + krknB1

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N. C. MELCHIOR AND J. R. MELCHIOR 610

This equation is compat,ible with the experimental results only if the term ~Jc~~R is negligible with respect to the other three groups of terms within the bracket.

Since no reaction pattern which involves the formation of appreciable amounts of Ez has been found to provide an equation compat,ible with the experimental findings, one can conclude that magnesium ion does not react with the active sit,e of the free enzyme, All four reaction mechanisms which are compatible with the experimental results indicate the formation of appreciable amounts of an enzyme-ATP4 complex, but differ in the role assigned to this complex. Since these four mechanisms give equations for V,/V, which are identical in form, it is not possible to distinguish between them by this kind of measurement.

Our results are compatible with those reported by Raaflaub and Leupin (13), although the considerations above show that their conclusion that the magnesium-ATP complex could not be the substrate for yeast hexo- kinase is not justified on kinetic evidence. The reason the experiments of these authors showed no dependence of V/V, on (ATP)T can be found by considering the terms in Equation 3. At all levels of (ATP)T used by them, the term CY is small with respect to other denominator terms and the equation approaches Vo/V, = C/(C + &4). Since C = 20AB, this is equivalent to V0/V, = 20B/(20B + /3), which is identical with the equation for an enzymic reaction in which B (in this case magnesium ion) is the substrate. This limiting form of Equation 3 predicts half maxima1 velocity at high (ATP)T when 20B = 0, that is when (Mg++) is 1.4 X 1O-2 mu. This corresponds to a “pMg” of 4.85 which is in excellent agree- ment with the half maximal velocity shown in Curve A of Fig. 2 in the paper of Raaflaub and Leupin (13). It might also be noted that if the association constant for the magnesium-ATP complex used in this paper were applied to the experimental data in Curve B, Fig. 2, in their paper, Curves A and B, Fig. 2, would be almost perfectly superimposed, as theory indicates they should be.

Ionic Strength Effects-The influence of ionic strength upon the velocity of the yeast hexokinase-catalyzed reaction between glucose and ATP (Fig. 8) should be understandable by means of Equation 3. This equa- tion quantitatively accounts for the measurements made at an ionic strength of 0.3 and it seems reasonable to assume, as a first approximation, that the form of the equation does not change with change in ionic strength. Equation 3 can then be written

00) VO UC v,” (Cjc + U + WAjzd

where fi is the activity coefficient of the ith ion and U and TV are the values which LY and p, respectively, would have at zero ionic strength.

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626 COMPLEX METAL IONS AND YEAST HEXOKINASE

The Debye-Hiickel equation

(11) -1ogfi = (constant) (Z?) (jb)

permits us to predict that in dilute solutions fA will decrease in value with increase in ionic strength more rapidly than fc. The effect of this change on the value of V,/V, will, however, depend upon the relative values of C and WA. If WA is large with respect to C [(ATP)* >> (Mg)T], the denominator of the fraction will decrease with increase in ionic strength much more rapidly than the numerator, with the result that V,/V,,, will increase markedly. On the other hand, if WA is negligible with respect to C [(ATP)T << (Mg)T], the numerator of the fraction will decrease in value slightly more rapidly than does the denominator (the term U does not change) and a slight, perhaps negligible, change in V,/V, is predicted. In other words, in dilute solution, VO should increase with ionic strength when (ATP)T is greater than (Mg)T, but there will be little effect when (ATP)T is less than or equal to (Mg)T. These predictions are in accord with the observations presented in Fig. 8.

At higher ionic strengths, a meaningful prediction is difficult because additional terms must be included in Equation Il. These terms include constants which must be determined separately for each ionic species, and are not at present known for ATP-4 or MgATPe2. From the known effects of increase of ionic strength on the activity coefficients of other polyvalent ions it is, however, possible to state that the inhibition observed at an ionic strength of 0.2 when (ATP)T exceeds (Mg)T is not inconsistent with this analysis.

E$ect of Sodium Ion-Increase in the sodium content of a reaction mix- ture at constant ionic strength causes a decrease in Vo which is not ac- counted for by the concomitant changes in MgATP-2 and ATPe4. This is shown in Fig. 7, where the experiment.al results are compared with V,/V, calculated from Equation 3. In fact, Equation 3 predicts an increase in V,/Vm for one of the experiments shown. One must conclude that there is a specific effect of one of the sodium-containing species pres- ent. To explore this situation further, Equation 3 was modified to include this unknown sodium species “NaX”

(12) VO c -E V7Pl Ic + a + E’A + A(Nax)l

This equation can be rearranged to

(13) CV, -

170 - C - a - &4 = A(NaX)

Values for the left side of Equation 13 obt.ained from the experiments

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N. C. MELCHIOR AND J. B. MELCHIOR 621

shown in Fig. 7 are plotted in Fig. 11 against the concentrations of the two sodium-containing molecular species known to be present in these solutions. The observed inhibition seems to be related to sodium ion rather than to the sodium-ATP complex, and one may tentatively conclude that there is significant interaction of Naf with at, least, one of the enzyme species present, but, no measurable interaction of NaATP-3 with the en- zyme.

These findings cause one to question Mechanisms I, II, and possibly III, since these mechanisms assume that the enzyme binds both MgATP-2 and ATPm4, and it, is difficult to accept these assumptions simultaneously with the failure to bind significant amounts of NaATP-3, which is inter-

0.2 0.4 0.1 0.2 0.3

(NAATP-~) (NA*) , M. FIG. 11. Identification of the sodium-containing species responsible for the in-

hibition of the yeast hexokinase reaction. Experimental data from Fig. 7; ordinate, left side of Equation 13. 0, (Mg)T = (ATPJT = 1.0 mnr; 0, (Mg)T = 0.5 mM; (ATP)T = 1.0 mM; (NaATP-a) is mM; (Na+) is M.

mediate in both charge and shape (8). The kinetic measurements ex- pressed in Equation 3 show no significant, binding of Mg++ at the active site of the free enzyme. Therefore binding of Na+ by the free enzyme is improbable and the observed inhibition of yeast, hexokinase by free Na+ is probably due to reaction of Na+ with the enzyme-ATP complex.

Inhibition of the yeast hexokinase reaction by ATP in excess of the magnesium present is a molecular mechanism by which the metabolism of a cell can be controlled in part by the products of metabolism, one of t.he molecular mechanisms for homeostasis. This might explain the ex- perimental findings of Lynen and Koenigsberger (22) that the rate of uptake of glucose by living yeast was approximately one-half as great in t,he presence of oxygen as in the absence of oxygen. -4s these authors pointed out, the increased amount of ATP expected in the presence of oxygen should, on simple kinetic reasoning, cause an increase in the rate of glucose phosphorylation and an increase in the uptake of glucose. Our

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622 COMPLEX METAL IONS AND YEAST HEXOKINASE

results are similar to those reported by Lardy and Parks (1‘2) for liver fructokinase and muscle phosphofructokinase. These authors pointed out the possible regulatory effect of the inhibition by ATP in excess of the available magnesium. It is important to remember that, while this regulatory mechanism is only one of many, it does exist. Evaluation of its importance relative to other regulators of metabolism in a given cell requires more information about cell metabolism than is now available.

SUMMARY

The value of quantitative knowledge of the equilibrium constants for the interaction of adenosine triphosphate (ATPm4) with various cations has been demonstrated by their use in the analysis of the effects of changes in a number of variables on the initial velocity of the yeast hexokinase- catalyzed reaction between glucose and ATP. An equation was developed which quantitatively accounted for the effects due to changes in the mag- nesium and ATP content of the reaction mixture. This equation provided a reasonable explanation for the effects of changes in ionic strength on the initial velocity; it made possible the demonstration that there was specific inhibition of yeast hexokinase by a sodium-containing species and provided suggestive evidence that this inhibitor was Naf rather than NaATPm3.

It has been shown that several different mechanisms for the formation of an enzyme-ATP-magnesium complex yield kinetic equations of identical form which are equally compatible with the experimental results. None of the acceptable mechanisms permits any appreciable formation of an enzyme-magnesium complex, but all require the formation of significant amounts of an enzyme-ATP complex.

BIBLIOGRAPHY

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Norten C. Melchior and Jacklyn B. MelchiorREACTION

IN THE YEAST HEXOKINASE THE ROLE OF COMPLEX METAL IONS

1958, 231:609-624.J. Biol. Chem. 

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