Kinetics Study of Yeast Pyruvate Kinase after Modification of Exposed

The reactivity and number of sulfhydryl groups of pyruvate kinase from baker’s yeast (Saccharomyces cereuisiae) are reported. The 20 total sulfhydryl groups (five per subunit) react as three different classes with 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB). The first class of four (one per subunit) react at 30°C and pH 7.5 with a second order rate constant of 2.56 X lo5 M-~ min-‘, which is 38 times more rapid than the reaction with reduced glutathione under similar conditions. The second class of eight (two per subunit) react with a rate of 9.2 M-’ min-‘. Denaturing conditions are required for the third class of eight (two per subunit) to react. Four (one per subunit) of the second class can be protected from reaction by kinetically saturating concentrations of KCl, MgClz, Fru-1,6-Pz, and P-enolpyruvate. Substi- tuting pyruvate or ADP for P-enolpyruvate or elimi- nating Fru-1,6-Pz from the reaction mixture destroyed the effect. The rates of the reaction with DTNB

The reactivity and number of sulfhydryl groups of pyruvate kinase from baker's yeast (Saccharomyces cereuisiae) are reported. The 20 total sulfhydryl groups (five per subunit) react as three different classes with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The first class of four (one per subunit) react at 30°C and pH 7.5 with a second order rate constant of 2.56 X lo5 M-~ min-', which is 38 times more rapid than the reaction with reduced glutathione under similar conditions. The second class of eight (two per subunit) react with a rate of 9.2 M-' min-'. Denaturing conditions are required for the third class of eight (two per subunit) to react. Four (one per subunit) of the second class can be protected from reaction by kinetically saturating concentrations of KCl, MgClz, Fru-1,6-Pz, and P-enolpyruvate. The sulfhydryl modification experiments are consistent with the hypothesis that the sulfhydryl residues are not directly involved in the catalysis, and that reaction of exposed sulfhydryl groups with DTNB results in a conformation with a lowered V,,,,, and an increased K,,,.
Pyruvate kinase, one of the key enzymes in the glycolytic pathway, catalyzes the transfer of a phosphoryl group from Penolpyruvate to ADP, yielding pyruvate and ATP. Yeast pyruvate kinase exhibits sigmoid v versus s curves when Penolpyruvate is varied, which become hyperbolic in the presence of Fru-1,6-Pz (1). The yeast enzyme, as well as those from other sources, exhibits hyperbolic v versus [ADP] curves, whether Fru-l&P2 is present or not (2-4). In 1971, Bondar and Suelter (5) showed that yeast pyruvate kinase from Saccharomyces cerevisiae contained 15 to 25 free sulfhydryl groups/mol, depending on the preparation. A biphasic inactivation by 5,5'dithiobis(2-nitrobenzoic acid) was also reported; that is, a slower inactivation was noted after loss of the first 50% of the original activity. Wieker and Hess (6) also examined the thiol reactivity of this enzyme from Saccharomyces carlsbergensis.
Their results differed somewhat from those reported for S. cerevisiae (5).
In order to clarify differences and to ascertain the functional role of sulfhydryl groups in yeast pyruvate kinase, we reinvestigated the sulfhydryl content of the enzyme from S. cerevisiae. The experiments provide evidence that the sulfhydryl groups of yeast pyruvate kinase do not directly participate in the catalytic function. Inactivation by DTNB,' or by other sulfhydryl reagents, appears to be a secondary effect due either to induced conformational changes or to a steric hindrance of substrate interaction or both. A preliminary report of this work has been given (7). '    d Activity too low to obtain meaningful kinetic data.
EXPERIMENTAL PROCEDURES The "Experimental Procedures," including materials and methods are described in the adjacent miniprint.'

RESULTS
The major portion of the "Results," including most figures and tables, is given in the adjacent miniprint.
Certain salient points are indicated in the parent section.
Number of Sulfhydryl Residues--In order to ensure that all sulfhydryl residues of yeast pyruvate kinase were in the reduced form before examining the DTNB reaction, the enzyme was incubated with 20 mM dithiothreitol prior to passage over Sephadex G-25 to remove salt and excess dithiothreitol (see "Experimental Procedures" in miniprint).
In the presence of denaturants, 6 M guanidine.HCl, or SDS, 20.5 f 0.33 sulfhydryl groups/m01 of enzyme reacted with DTNB (Fig.  IS). In the absence of denaturants, 12.1 -t 0.83 sulfhydryl residues reacted (Fig. IS). Similar results were obtained by reaction with pMB, indicating that 12 of the 20 sulfhydryl residues in yeast pyruvate kinase were exposed.
The sulfhydryl groups of the native enzyme could be grouped into three classes according to their reactivity toward DTNB. Four of the 12 exposed residues reacted during the mixing of reactants  (Table IIS). The second class of 8 remaining exposed residues reacted with a rate constant of 9.2 Mm' s-' at 30°C and pH 7.5. This class of 8 sulfhydryl residues was shown to have a pK = 9.54 (Fig. 2s). The 8 sulfhydryl residues that reacted only under denaturing conditions constitute the third class. Properties of the Modified Enzyme-The inactivation of enzyme in the presence of excess DTNB was first order and gave the same rate (0.19 min-') whether or not the catalytic activity of the enzyme at various times was measured in the presence or absence of Fru-1,6-Ps (Fig. 3s). This rate was identical with the rate observed for reaction of the second class of eight exposed sulfhydryl groups with DTNB, showing that the changes in activity correlate directly with modification of the second class of sulfhydryl residues. The DTNB modification was reversible since 92% or 82% of the original enzyme activity was obtained when assayed with or without Fru-1,6-Pz 30 min after addition of dithiothreitol (Fig. 3s). Addition of excess NaCN to the DTNB-modified native enzyme resulted in the incorporation of 12 mol of cyanide/m01 of enzyme (Fig. 4s) with a partial recovery of catalytic activity (Fig. 3s).
Substrate Protection-Addition of KCl, MgC12, P-enolpyruvate, and Fru-1,6-P? provided protection for 4 sulfhydryl residues from reacting with DTNB. No other combination of substrate or effecters provided protection.
Kinetic Properties of the Modified Enzyme-The kinetic properties, including the K, for the two substrates, P-enolpyruvate and ADP, in the presence and absence of the allosteric effector, Fru-1,6-Pz, and the K, for the effector were determined for four enzyme derivatives. The results are compared with those of the unmodified enzyme in Table I. The enzyme derivative containing 12 TNB residues (PK-TNB12) was not stable, and thus it was not possible to obtain a reproducible V,,,,,. However, when experiments were completed 4 and 24 h after initiation of the DTNB modification, V,,,,, for the 24-h enzyme was about one-half that of the 4-h enzyme, but the K,,, for P-enolpyruvate was identical. Whatever the origin of the inactivation, it was important to note that it does not result in an altered K,. The K, for Penolpyruvate for PK-TNB12 was over lo-fold larger than that for native enzyme when assayed in the presence of 1 mM Fru-1,6-Pz. Insufficient activity was obtained in the absence of Fru-1,6-Pz to warrant study. The Hill slope (no) (8) for Penolpyruvate saturation in 1 mM Fru-1,6-P:! for native enzyme is equal to 1, while that for PK-TNB12 was greater than 1, suggesting that 1 mM Fru-1,6-Pz was no longer saturating. This was confiied as indicated in Table I; a K, of 0.25 mM for Fru-1,6-Pz was obtained for PK-TNB12, which is nearly 20fold higher than that observed for native enzyme. The K, for ADP was nearly 6-fold larger than that for native enzyme. The Hill slope for ADP was always 1.0.
The enzyme with 8 TNB residues/m01 (PK-TNBs) exhibited 80 to 90% of the maximum velocity of the native enzyme regardless of whether or not the assay was completed with Fru-1,6-Pz. The ratio, v (+ Fru-1,6-Pn)/V (-Fru-1,6-P*) was in the range of 1.1 to 1.4. The K, for P-enolpyruvate was identical with that obtained with native enzyme, while that for ADP was elevated nearly g-fold. The K, for Fru-1,6-Pz determined with 10 mM P-enolpyruvate was 20 pm, which is nearly identical with that observed with native enzyme.
The interesting feature of the derivative with I2 cyano groups/mol is that the ratio of the activity with and without Fru-1,6-Pz is between 2 and 3. The maximum velocity of this derivative is greater than that found with PK-TNB12, indicating that the CN residue at the site which is blocked by Penolpyruvate has a smaller effect than TNB on the turnover number. The K, for ADP and P-enolpyruvate, whether measured in the presence or absence of Fru-1,6-Pz, was always larger than that obtained with native enzyme, but smaller than that of PK-TNB1,.
The K, for Fru-1,6-Pn was also increased, but the increase was much smaller than that observed with PK-TNB12.
The reduced enzyme (PK-SH,), prepared by reduction of PK-TNB1:! with dithiothreitol, had a ratio of maximum velocities plus or minus Fru-1,6-Pz of 1.05. As expected, all of the kinetic parameters except the maximum velocity of the PK-SH, were identical with those of the native enzyme.