Equilibrium Isotope Exchange Kinetics Research Articles

L-Aspartate Association Contributes to Rate Limitation and Induction of the T → R Transition in Escherichia coli Aspartate Transcarbamoylase: EQUILIBRIUM EXCHANGES AND KINETIC ISOTOPE EFFECTS WITH A Vmax -ENHANCED MUTANT, Asp-236 → Ala

Equilibrium isotope exchange kinetics (EIEK) and kinetic isotope effects have been used to determine the mechanistic basis for the altered kinetic characteristics of a mutant version of Escherichia coli aspartate transcarbamylase in which Asp-236 of the catalytic chain is replaced by alanine (Asp-236-->Ala). The [14C]Asp<--> N-carbamyl-L-aspartate (CAsp) and [14C]CP<-->CAsp exchange rates, observed as a function of various reactant-product pairs, exhibited dramatic increases in maximal rates, along with decreases in substrate half-saturation values and cooperativity. The carbon kinetic isotope effect, 13C versus 12C at the carbonyl group of carbamoyl phosphate, for the Asp-236-->Ala enzyme decreased toward unity as [Asp] increased, as observed for the wild-type enzyme. Both the kinetic isotope effects and EIEK results indicate that the Asp-236-->Ala enzyme operates by the same ordered kinetic mechanism as the wild-type enzyme. Although activation effects by ATP and N-phosphonacetyl-L-aspartate are lost, inhibition by CTP was apparent in equilibrium exchanges. Simulation of the EIEK data indicated that the best fit to the observed changes in saturation curves was obtained by preferentially increasing the rate of the T-->R transition, kappa T-->R, thereby destabilizing the T-state and increasing the equilbrium constant for the T<-->R transition. A multistep model for Asp bindng to aspartate transcarbamoylase is proposed, in which Asp induces the initial conformational changes that in turn trigger the T-->R transition, followed by stepwise filling of the remaining active sites.

17] Equilibrium isotope exchange in enzyme catalysis

In 1980, Purich and Allison observed that the equilibrium isotope exchange kinetics (EIEK) method was “undergoing rapid expansion in theory and experiment.” At that time, in the absence of convenient methods for modeling enzyme kinetic behavior at equilibrium, the theoretical base for EIEK was badly underdeveloped compared to what existed for initial velocity and other methods. With the development of the ISOBI and ISOMOD programs, described here, many of these problems have been overcome. The primary objective of this chapter is to explain the breakthroughs in the methodology, resulting from the development of computer programs for quantitative simulation and analysis of EIEK data. These new approaches offer the nonexpert instant access to a versatile theoretical basis for almost any complex Bi Bi or Ter Ter kinetic scheme, unfettered by the requirement to derive complex algebraic equations in many cases an impossible task. Without these new methods, EIEK data analysis would still be done by the qualitative logic set forth by Boyer and Silverstein in the early 1960s.

Kinetic and regulatory mechanisms for (Escherichia coli) homoserine dehydrogenase-I. Equilibrium isotope exchange kinetics.

Isotope exchange kinetics at chemical equilibrium were used to probe the mechanisms of substrate binding and regulatory behavior of homoserine dehydrogenase-I from Escherichia coli. At pH 9.0, 37 degrees C, Keq = 100 (+/- 20) for the catalyzed reaction: L-aspartate-beta-semialdehyde + NADPH + H+ = L-homoserine + NADP+. Saturation curves for the exchange reactions, [14C]L-homoserine <--> L-aspartate-beta-semialdehyde and [3H]NADP+ <--> NADPH were observed as a function of different reactant-product pairs, varied in constant ratio at equilibrium. The NADP+ <--> NADPH exchange rate was inhibited upon variation of pairs involving L-aspartate-beta-semialdehyde and L-homoserine, consistent with preferred order random binding of cofactors before amino acids. Optimal rate constants, derived by simulations of equilibrium isotope exchange kinetics data with the ISOBI program, indicate faster dissociation of amino acids than cofactors from the central complexes but nearly equal rates for association of cofactors and amino acids to free enzyme. Rate limitation of net turnover in both directions is determined by dissociation of cofactor from the E-cofactor complex. The allosteric modifier, L-threonine, produces distinctive perturbations of the saturation curves for isotope exchange, which were analyzed systematically with the ISOBI program. The best fit to the data was obtained by L-threonine inhibiting catalysis between the central complexes without altering substrate association-dissociation rates. Simulations also showed that rate-limiting catalysis suppresses the kinetic inhibition effects that are characteristic of preferred order substrate binding, producing patterns typical for a (rapid equilibrium) random kinetic scheme.

Homoserine Dehydrogenase-I ( Escherichia coli): Action of Monovalent Ions on Catalysis and Substrate Association-Dissociation

Changes in the kinetic properties of homoserine dehydrogenase-I (HD-I) from Escherichia coli, caused by substitution of Na + for the normal activating monovalent ion, K +, has been investigated by equilibrium isotope exchange kinetics (EIEK). HD-I, part of the aspartokinase/homoserine dehydrogenase-I complex, is one of the few dehydrogenases to exhibit allosteric feedback regulation and cation activation. EIEK methods are especially useful for definitively identifying which rate constants are altered by bound modifiers. Saturation curves for the [ 14C]Hse ⇄ ASA and [ 3H]NADP + ⇄ NADPH exchanges were compared in the presence of K + vs Na +, varying different combinations of substrate pairs in constant ratio at equilibrium. Kinetic differences between the K + and Na + enzymes were analyzed systematically by simulations with the ISOBI program. This analysis clearly demonstrates that substituting Na + for K + shifts the kinetic mechanism from preferred order random to a nearly random order scheme, along with causing significant rate limitation at catalysis between the central complexes. Initial velocity kinetics demonstrate that HD-I has a 10-fold higher affinity for Na + than K +, but that the Nat-enzyme is 10-fold less active and exhibits higher substrate K m values, especially for L-Hse.

Preferred order random kinetic mechanism for homoserine dehydrogenase of Escherichia coli (Thr-sensitive) aspartokinase/homoserine dehydrogenase-I: equilibrium isotope exchange kinetics.

Isotope exchange kinetics at chemical equilibrium have been used to investigate the kinetic mechanism of homoserine dehydrogenase (EC 1.1.1.3) of the (Thr-sensitive) aspartokinase/homoserine dehydrogenase-I multifunctional enzyme from E. coli. For the reaction (L-ASA + NADPH + H+ = L-Hse + NADP+), at pH 9.0, 37 degrees C, Keq = 100 (+/- 20). Under these conditions, the rate for exchange of [14C]-L-homoserine (Hse) in equilibrium L-aspartate-beta-semialdehyde (ASA) is nearly twice that for the [3H]-NADP+ in equilibrium NADPH exchange. This indicates that covalent interconversion between reactants and products bound in the active site cannot be rate-limiting. Upon variation of the concentrations of all four substrates in constant ratio at equilibrium (to minimize dead-end complex formation), the Hse in equilibrium ASA exchange increased smoothly toward a maximum. In contrast, the NADP+ in equilibrium NADPH exchange rate increased to a maximum value at partial saturation, then decreased to approximately half the maximum rate. These data are consistent with a preferred-order random kinetic mechanism in which the dominant pathway involves association of NADPH prior to L-ASA and dissociation of L-Hse prior to NADP+.

Kinetic consequences of site-specific mutation of Glu-239 → Gln in E. coli aspartate transcarbamylase: comparison with catalytic subunits and Phe-240 mutant enzyme

The kinetic characteristics of E. coli aspartate transcarbamylase, altered by site-specific mutagenesis of Glu-239 → Gln, have been determined by equilibrium isotope-exchange kinetics and compared to the wild-type system. In wild-type enzyme, residue Glu-239 helps to stabilize the T-state structure by multiple bonding interactions with Tyr-165 and Lys-164 across the c 1-c 4 subunit interface; upon conversion to the R-state, these bonds are re-formed within c-chains. Catalysis of both the [ 14C] Asp⇌C-Asp and [ 32P]ATP ⇌ P 1 exchanges by mutant enzyme occurs at rates comparable to those for wild-type enzyme. Saturation with different reactant/product pairs produced kinetic patterns consistent with strongly preferred order binding of carbamyl-P prior to Asp and carbamyl-Asp release before P 1. The kinetics for the Gln-239 mutant enzyme resemble those observed for catalytic subunits (c 3), namely a R-state enzyme (Hill coefficient n H = 1.0) and K m ( Asp) ≊ 6 mM . The Glu-239 → Gln mutation appear to destabilize both the T- and R-states, whereas the Tyr-240 → Phe mutation destabilizes only the T-state.

Effectors of Escherichia coli aspartate transcarbamoylase differentially perturb aspartate binding rather than the T-R transition.

New systematic methods developed for equilibrium isotope exchange kinetics have been used to analyze the effects of activator ATP and inhibitor CTP with Escherichia coli aspartate transcarbamoylase. This indepth approach requires (a) variation of [modifier] with fixed subsaturating levels of substrates, and (b) variation of at least three combinations of reactant-product pairs in constant ratio at equilibrium: [A,B,P,Q], [A,P], and [B,Q] with the co-substrates held constant, in the presence and absence of added modifier. Both ATP and CTP had much stronger effects on the [14C]Asp in equilibrium C-Asp exchange rate than on [32P]C-P in equilibrium Pi. The bisubstrate analog N-phosphonacetyl-L-aspartate activated, then inhibited, Asp in equilibrium C-Asp more strongly than C-P in equilibrium Pi. N-Phosphonacetyl-L-aspartate gave complete (100%) inhibition, whereas CTP inhibition of either exchange was only partial. Substrate saturation curves in the presence and absence of effectors indicate that ATP and CTP perturb the observed values of Rmax and S0.5 in different fashions without appreciably changing the observed Hill number. Computer simulations indicate that the primary site of ATP and CTP action is the association rate for Asp, not the allosteric T-R transition. This finding is substantiated by previous studies in which modified aspartate transcarbamoylase had lost cooperative Asp binding without loss of sensitivity to effectors, or in which sensitivity to one effector could be deleted selectively. The present results, with newly devised computer simulation and analysis methods, illustrate the usefulness of equilibrium isotope exchange kinetic probes for providing unique insights to enzyme regulatory mechanisms, to define exactly which steps are altered in a given kinetic mechanism.

Kinetic mechanism of native Escherichia coli aspartate transcarbamylase

Equilibrium isotope exchange kinetics have been used to reinvestigate the kinetic mechanism of Escherichia coli aspartate transcarbamylase (aspartate carbamoyltransferase) at pH 7.0, 30 °C. K eq = 5.9 (±0.6) × 10 3, allowing variation of substrate concentrations above and below their K m values in all experiments, a condition not possible at pH 7.8 [ F. C. Wedler and F. J. Gasser (1974) Arch. Biochem. Biophys. 163, 57–68 ]. The rate of the [ 14C]Asp ⇌ N-carbamoyl l-aspartate (CAsp) exchange reaction was five times faster than that of [ 32P]carbamyl phosphate (CP) ⇌ P i , which argues strongly against the rapid equilibrium random mechanism previously proposed by E. Heyde, A. Nagabhushanam, and J. F. Morrison [ Biochemistry 12, 4718–4726 (1973) ]. Substrate concentrations were varied either as reactant-product pairs (holding the other pair constant) or together simultaneously in constant ratio at equilibrium. The resulting kinetic saturation patterns were most consistent with a preferred order random kinetic mechanism, with CP binding prior to Asp and with CAsp being released before P i. Weak inhibition effects at high substrate levels could be accounted for by multiple weak dead-end complexes or ionic strength effects. Computer-based simulations have led to a set of rate constants that fit the experimental data, are in agreement with rate constants measured previously by pre-steady-state methods, and predict the correct initial velocities in the forward and reverse directions. Simulations also show that rate constants consistent with any of the various alternative mechanisms do not provide good fit to the experimental data. A model for the kinetic mechanism is considered, in which the binding of Asp prior to CP may restrict access of CP to the active site, but CP binding prior to Asp potentiates the enzyme for the allosteric (T-R) transition, centered entirely upon the Asp binding process.

33] Isotope exchange studies of regulatory enzymes: “Fingerprint” kinetic patterns for modifier action

This chapter discusses the isotope exchange studies of regulatory enzymes, a fingerprint kinetic patterns for modifier action. Equilibrium isotope-exchange kinetics can provide unique insights to the modes of action of enzyme-bound activators or inhibitors, since one can observe both rate-limiting and non-rate-limiting steps in overall catalysis. Ten different theoretical modes of modifier action are simulated and analyzed by computer for the two substrate-two product system, A + B = P + Q. Kinetic patterns were derived for both random and compulsory order substrate binding systems, using known equations for exchange rates R(exchange rates) (A⇋P) and R' (B⇋Q). Complete elucidation of modifier action requires that one: (a) Determine the order of substrate binding by varying the concentration of all appropriate substrate-product pairs in constant ratio at equilibrium. (b) Repeat (a) in the presence of added modifier, then analyze the perturbations of R and R' with double-reciprocal plots. (c) Observe the effect on R and R' of varying [modifier], holding [substrates] constant at ≈3K m of each. These procedures constitute and result in four different experiments from which the perturbations because of added modifier can be calculated and graphed. The model studies indicate that one obtains a unique set of kinetic patterns a fingerprint for each mode of activation or inhibition. New insights to the intimate details of the regulation of many key enzymes are now possible on a more systematic basis by these techniques.

Mechanisms of Substrate Binding with Glutamine Synthetase: EQUILIBRIUM ISOTOPE EXCHANGES WITH THE OVINE BRAIN, PEA SEED, AND ESCHERICHIA COLI ENZYMES

Abstract Equilibrium isotope exchange kinetics were used to investigate the sequences of substrate binding with ovine brain, pea seed, and Escherichia coli glutamine synthetases. Without exception, the relative rates of exchange are (glutamate ⇆ glutamine) g (NH3 ⇆ glutamine) g (Pi ⇆ ATP) ≃ (ADP ⇆ ATP). This suggests that the rate of net turnover at saturating substrate levels is limited more strongly by the rate of nucleotide release than by the rate of covalent interconversion. With the ovine brain enzyme, the kinetic patterns of equilibrium exchange are consistent with a partially ordered sequence of substrate binding, where ATP binds before NH3, glutamine leaves before ADP, but glutamate and phosphate are bound and released randomly on their respective sides of the reaction. This particular binding order does not permit observation of those partial exchange reactions expected if an enzyme-bound γ-glutamyl phosphate intermediate were formed. Upon variation of the concentration of all substrates in constant ratio at equilibrium, the pea seed, the Mn2+-activated adenylylated and the Mg2+-activated unadenylylated E. coli enzymes exhibit exchange kinetic patterns consistent with random substrate addition and release. The kinetic data also suggest synergism of substrate binding between glutamate and ATP. A brief section on theory and interpretation of kinetic patterns of equilibrium exchanges as related to substrate-binding order is presented.