[Kinetic manifestations of slow conformation rearrangements of lactate dehydrogenase. An experiment and a mathematical model]. / Issledovanie kineticheskikh proiavlenii medlennykh konformatsionnykh perestroek v laktatdegidrogenaze. Eksperiment i matematicheskaia model'.

Non-steady-state kinetics of lactate dehydrogenase (LDH) catalyzed reaction was investigated for a wide time interval (from 100 msec to 1-3 min) by using stopped-flow methods. A two-stage character of LDH reaction, slow changes like a lag-period on kinetic curves at pH 8.0, flexions on kinetic curves after pre-mixing LDH with NAD+ and pyruvate have been revealed. The graph theory for mathematical analysis of experimental data was applied, which has been developed for the non-steady-state kinetics. An enzyme model of the two-conformer LDH structure was used. The reaction scheme with a preferential inhibition of one of the conformers (pH 8.0) is suggested. The obtained values of kinetic constants prove that transitions between LDH conformers must be slow.

[A model of oligomeric enzymes explaining stepwise kinetic curves]. / Model' oligomernykh fermentov, ob''iasniaiushchaia "stupenchatye" kineticheskie krivye.

The complex kinetics of some oligomeric enzymes has been analysed. For interpreting such kinetics a theoretical model of an oligomeric enzyme is suggested, which is a modification of the "flip-flop" mechanism considered by Lazdunski et al. in 1971. Using our model, one can qualitatively interpret stepwise kinetic curves, i.e. the curves with few intermediate plateaus. Such curves are known from literature but have not exhaustively been explained. It is supposed that the enzyme kinetic curves can sometimes be of non-differential functions. The model used can also be applied to the kinetics of polyenzyme complexes.

[Effect of anions on inhibition of lactate dehydrogenase by pyruvate]. / Vliianie anionov na ingibirovanie laktatdegidrogenazy piruvatom.

The effect of anions Cl- and I- on structural and kinetic properties of LDH was investigated. It was shown that anions are specific inhibitors of LDH competing with pyruvate in the active ternary complex, LDHNADHpyq. The following dissociation constants for the anions were obtained from inhibition data: 0.4 +/- 0.02 and 0.07 +/- 0.01 M for Cl- and I-, respectively. The slope of Hill plot are near 1.0. The anions abolished the inhibition of LDH at high pyruvate concentrations. The following dissociation constants were obtained from these data: 0.1 and 0.015 M for Cl- and I- respectively. The inhibition by anions and the abolishing of substrate inhibition by anions were studied also for the lactate oxidation reaction. The dissociation constants for anions obtained from these data are in good correlation with the constants obtained for the pyruvate reduction reaction. It was concluded that anions do not interact with the group at the catalytic site with pK approximately 7.8, presumably His-195. The degree of pyruvate inhibition does not depend on the buffer system. The differences in the degree of inhibition obtained previously in phosphate, imidazole and tris-buffer systems can be explained by the presence of Cl- anions in the last two buffer. The rate constants of hydroxy leads to keto pyruvate transition was obtained in various buffer systems. It was shown that the hydroxy-form of pyruvate does not cause the inhibition of LDH.

[Possible nature of negative cooperation in D-glyceraldehyde-3-phosphate dehydrogenase]. / O vozmozhnoi prirode otritsatel'noi kooperativnosti v D-glitseral'degid-3-fosfatdegidrogenaze.

Some considerations concerning the detailed mechanism of negative cooperativity in GPD are proposed. The hypothesis represents a modification of the sequential model (Koshland et al.) taking into account last experimental data about the binding of NAD analogs and fragments. Two main facts have been used as a basis for the model: 1. Neither ADP-ribose nor nicotinamide mononucleotide (NMN) fragments of NAD show negative cooperative binding to GPD. 2. Neither modifications of adenine and nicotinamide part of NAD (epsilon-NAD, hypoxantine-NAD, oxidized and reduced-NAD) nor enzyme modifications by various reagents acting in the catalytic site affect considerably the cooperativity of coenzyme binding although the affinity between enzyme and coenzyme (analogs) substantially changes depending on the nature of modification. Probably the structural integrity of a coenzyme molecule is necessary for the cooperative binding to GPD. On the other hand, numerous modification studies can be interpreted as proving the absence of direct participation of adenine and nicotinamide rings in the mechanism of negative interactions between NAD-binding sites. It appears reasonable to assume that direct or indirect interactions of riboseAD and pyrophosphate groups of NAD with the "loop" of adjacent subunit might be necessary for the tight coenzyme binding to the first active site of the r-dimer(s) symmetric across the R-axis. After the tight binding of the first NAD molecule on r-dimer with the "loop" participation, the symmetrical movement of second "loop" might be highly restricted. It was postulated that only asymmetric conformational transition is possible in contact areas between subunits across the R-axis. Such asymmetric rearrangement can explain the nonequivalent binding of NAD to a prior symmetric dimmer(s).

[pH-dependence of the structural and functional properties of lactate dehydrogenase (M4). Indirect cooperativity in lactate dehydrogenase]. / Issledovanie pH-zavisimosti strukturnykh i funktsional'nykh svoistv laktatdegidrogenazy (M4). O kosvennoi kooperativnosti v laktatdegidrogenaze.

The dependence of structural and functional properties of LDH on pH in the 6.0--9.0 region was investigated. There were no marked deviations of pyruvate reduction initial velocity curves from the Michaelis--Menten equation in a wide range of pyruvate concentrations. It was shown that Vmax changes negligibly in the 6.0--9.0 pH regions, but Km increased markedly with pH elevation. The pK value of 7.8+/-0.1 was obtained for 50% changes of pyruvate binding. The dependence of enzyme inhibition from pH at a high pyruvate concentration (20 mM) was investigated. At pH values above 8.0 pyruvate inhibition disappeared. The dependence of the inhibition degree from pH was estimated as pK 7.8+/-0.1. Hill coefficient (n) calculated from the curves of Km and the degree of substrate inhibition depending on pH was 1.6; n for pyruvate inhibition at pH 7.5 was 2 greater than n greater than 1 for moderate substrate concentrations (1--5 mM) and n approximately 1 for higher concentrations (5--40 mM). The value of n approximately 1 at pH 7.8 was obtained. The model suiting all available data concerning the cooperativity phenomena in LDH during protonation and inhibition by pyruvate is outlined. The model is based on the results indicating the slow isomerisation of LDH in ternary complexes with NADH and pyruvate and the absence of equilibrium on the intermediate stage of reaction.

[Kinetic manifestations of the interaction of active centers in swine skeletal muscle D-glyceraldehyde-3-phosphate dehydrogenase]. / Kineticheskie proiavleniia vzaimodeistvii aktivnykh tsentrov v D-glitseral'degid-3-fosfatdegidrogenaze iz skeletnykh myshts svin'i

The kinetic method and selective chemical modification have been used in studies of the kinetic manifestations of active site interactions in D-glyceraldehyde-3-phosphate dehydrogenase (GAP dehydrogenase). The reactions of glyceraldehyde and glyceraldehyde-3-phosphate oxidation were studied in the absence of substrate excess. In support of the data obtained previously it was shown that only a part of the tightly bound NAD molecules can be reduced after substrate addition. "Partial reducibility" is observed at various degrees of saturation of the enzyme with NAD involving a single NAD molecule per tetrametric enzyme. These facts can hardly be explained by assumption of functional non-equivalence of active sites, whether induced by coenzyme or preexisting in the apoenzyme. It was proven by selective alkylation of the catalytic SH groups that "partial reducibility" is due to the circumstance that equilibrium in the system under investigation is established at nearly equal NAD and NADH concentrations. A plot of initial reaction rates versus NAD concentration (at non-saturating substrate concentrations) gives S-shaped curves; this is explained by considerable enzyme activation upon saturation of the fourth site with coenzyme. After modification of three active sites with iodoacetate the S-shape of the curve disappeared. This fact leads to the conclusion that active site interactions are required for formation of the S-shaped curves. The activity of a single site functioning in the modified enzyme reached values equal to those of the active sites in the native enzyme in the fully activated state. A model is proposed which can explaine the variations in mode of enzyme activation in the native and modified states. It is suggested that the surroundings of all four SH groups must be altered in order to activate the enzyme; such changes can be induced either by alkylation of the SH groups or by NAD binding. Evidence is presented that important functional properties of GAP dehydrogenase cannot be elucidated at low enzyme concentrations and with excess of substrates: three active sites are saturated under such conditons and practically inactive, and the fourth site obeys Michaelis - Menten kinetics.