Isoleucyl-tRNA synthetase from Baker's yeast. Catalytic mechanism, 2',3'-specificity and fidelity in aminoacylation of tRNAIle with isoleucine and valine investigated with initial-rate kinetics using analogs of tRNA, ATP and amino acids

The aminoacylation of three modified tRNAIle species with isoleucine and with valine by isoleucyl-tRNA synthetase has been investigated by initial rate kinetics. For aminoacylation of tRNAIle-C-C-3'dA with isoleucine, a bi-bi uni-uni ping-pong mechanism has been found by bisubstrate kinetics and inhibition by products and by 3'dATP; for aminoacylation with valine a bi-uni uni-bi ping-pong mechanism. For isoleucylation of tRNAIle-C-C-A(3'NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni-bi uni-uni-uni ping-pong mechanism; for valylation of this tRNA a bi-bi uni-uni ping-pong mechanism is observed by bisubstrate kinetics and product inhibition. tRNAIle-C-C-2'dA was aminoacylated under modified conditions with isoleucine in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P[NH]P as product analog and by isoleucinol. Aminoacylation with valine is achieved in a rapid-equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3'dATP and valinol. All six reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle-C-C-A. The Km values of the three substrates and the kcat values of the six reactions are given. For aminoacylation at the terminal 2'OH group of the tRNA differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine have been calculated which result in discrimination factors of 181 and 167. For aminoacylation at the terminal 3'-OH group a difference of only 4.43 kJ and a low discrimination factor of only 6 is observed. Thus maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2'-OH group and conclusions drawn from experiments with modified tRNAs concerning 2',3'-specificity have led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNAs. The stability of Ile-tRNAIle-C-C-2'dA and Val-tRNAIle-C-C-2'dA, the lesser stability of Val-tRNAVal-C-C-2'dA and the instability of Thr-tRNAVal-C-C-2'dA are consistent with postulations for a 'pre-transfer' proofreading step for isoleucyl-tRNA synthetase and a 'post-transfer' hydrolytic editing step for valyl-tRNA synthetase at the terminal 3'OH group of the tRNA.     

A generalized theoretical treatment of the kinetics of an enzyme-catalysed reaction in the presence of an unstable irreversible modifier

A generalized theoretical treatment of the kinetics of an enzyme-catalysed reaction in the presence of an unstable irreversible inhibitor (or activator) is presented. Analytical expressions describing the time-dependence of product formation have been derived in coefficient form amenable to non-linear regression analysis for two operationally distinct types of reaction mechanism dependent on whether the reaction of the unstable modifier (X) with either or both the free enzyme (E) and enzyme-substrate complex (ES) occurs as a simple bimolecular process, or proceeds through the intermediacy of either or both adsorptive enzyme-modifier (EX) and enzyme-modifier-substrate (EXS) complexes in what may be considered as an extension of the Botts-Morales general modifier mechanism for (stable) reversible enzyme inhibitors and activators. Special cases of both models are classified in an analogous way to the traditional naming of reversible enzyme modifications, and guidelines concerning tests of mechanism and determination of kinetic parameters are given. In particular, it has been shown that kinetic constants describing enzyme inactivation by an unstable site-specific inhibitor forming a reversible EX complex prior to covalent modification step may be determined from a single progress curve. Kinetic analysis of the extended Botts-Morales mechanism describing irreversible enzyme inactivation has demonstrated that analytical expressions describing the time-course of product formation may be derived for a stable modifier by retaining the usual steady-state assumptions regarding the fluxes around ES and EXS provided quasi-equilibrium modifier binding to E and ES is assumed, but for unstable modifiers all of the binding steps must be assumed to be at quasi-equilibrium in the steady-state, except under restrictive circumstances.     

Quasi-equilibrium assumption in enzyme kinetics. Necessary and sufficient conditions and accuracy of its application for single-substrate reactions

Steady-state kinetics of compulsory-ordered single-substrate irreversible and reversible enzyme reactions with two, three, and arbitrary number of intermediates were observed. Necessary and sufficient conditions for application of the quasi-equilibrium assumption and restrictions of this assumption were found in cases of two and three intermediates in the equilibrium segment. For all cases, accuracy of the quasi-equilibrium assumption was evaluated.     

Quasi-equilibrium assumption for arbitrary mechanism of enzymatic reaction. criteria for existence of equilibrium segments

The possible application of the quasi-equilibrium assumption for an arbitrary mechanism of enzymatic reaction is considered. It is shown at what ratios of kinetic constants a segment consisting of two, three, and four intermediates may be considered as an equilibrium one. Expressions for evaluation of accuracy of distribution of intermediate concentrations inside the equilibrium segment and accuracy of determination of intermediate concentrations inside and outside the equilibrium segment as a function of the ratio of kinetic constants are derived. A method for determination of the limitations on the ratio of rate constants for an equilibrium segment of arbitrary structure is suggested.     

Kinetic characteristics of thiamine diphosphate biosynthesis by thiamine pyrophosphokinase from rat liver]

The kinetic analysis of bisubstrate enzymatic reaction catalysed by electrophoretically homogenous thiamine pyrophosphokinase (EC 2.7.6.2), isolated from rat liver has been carried out. Kinetic studies of the initial rates in the absence of the products and inhibition by the reaction products as well as the data from the equilibrium dialysis suggest that the reaction proceeds through the formation of a ternary enzyme-substrate complex. The combination with substrates and release of the products appears to be highly ordered. A possible scheme of the reaction mechanism is discussed.     

Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC)

Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-d-myo-inosityl-2-(N-acetyl-l-cysteinyl)amido-2-deoxy-alpha-d-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-MshD. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady-state and pre-steady-state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady-state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s-1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5'-O-[N-(l-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of approximately 306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of approximately 9.4 and approximately 5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.     

Crystallographic snapshots of the complete catalytic cycle of the unregulated aspartate transcarbamoylase from Bacillus subtilis

Here, we report high-resolution X-ray structures of Bacillus subtilis aspartate transcarbamoylase (ATCase), an enzyme that catalyzes one of the first reactions in pyrimidine nucleotide biosynthesis. Structures of the enzyme have been determined in the absence of ligands, in the presence of the substrate carbamoyl phosphate, and in the presence of the bisubstrate/transition state analog N-phosphonacetyl-l-aspartate. Combining the structural data with in silico docking and electrostatic calculations, we have been able to visualize each step in the catalytic cycle of ATCase, from the ordered binding of the substrates, to the formation and decomposition of the tetrahedral intermediate, to the ordered release of the products from the active site. Analysis of the conformational changes associated with these steps provides a rationale for the lack of cooperativity in trimeric ATCases that do not possess regulatory subunits.     

Action of lipolytical enzymes in biphasic organic-aqueous systems: dynamics of the irreversible Michaelis-Menten reaction

Through simple model analysis, the mass action kinetic model for lipolytic enzymes in biphasic aqueous-organic systems can be simplified using the quasi-steady state assumption (or the quasi-equilibrium state assumption) for the adsorbed enzyme E* or the enzyme-substrate complex E*S. Some parameter combinations leading to the above assumptions are derived confirmed by full numerical integration of the whole enzymatic process. The results may be classified into three categories: (1) the quasi-equilibrium state assumption for E*, (2) the quasi-steady state assumption for E*, and (3) the quasi-steady state assumption for E*S. Further simplification for both E* and E*S is also discussed. (c) 1993 Wiley & Sons, Inc.     

ATP-conjugated peptide inhibitors for calmodulin-dependent protein kinase II

Substrate analog peptides of CaMKII with varying degrees of the inhibitory potency were linked to ATPgammaS either by considering a phosphoryl transfer mechanism or simply by using a relatively long flexible linker. The latter bisubstrate inhibitors showed relatively little effects while the former ones improved inhibitory potency to different levels depending on the binding affinities of the peptide moieties. One of the mechanism-based bisubstrate inhibitors was then utilized to demonstrate an ATP-competitive but peptide substrate-uncompetitive inhibition, supporting an ordered binding mechanism for CaMKII.     

A general method for the analysis of random bisubstrate enzyme mechanisms

In the present communication, a general method for the kinetic analysis of random bisubstrate mechanisms is described. The method comprises a stepwise application of the following kinetic and ligand-binding experiments: determination of steady-state kinetic constants, product inhibition patterns, maximum rate relationships, application of alternate substrates, application of dead-end inhibitors, direct binding of substrates, kinetic isotope effects, and isotope exchange studies. This general method was applied to a practical example: a yeast alcohol dehydrogenase-catalyzed oxidation of 2-propanol by NAD⁺ at pH 7.0, 25°C. It was found that this fully reversible reaction proceeds by a steady-state random Bi-Bi mechanism, whereby both dead-end complexes are formed.     

A 70-amino acid zinc-binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer.

Interaction between a 70-amino acid and zinc-binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady-state kinetics, heavy-atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain-C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased "stickiness" of carbamoyl phosphate to the Zn domain-C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain-C trimer complex was about 10(-2) that from C trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deriving the rate equations for product inhibition patterns in bisubstrate enzyme reactions

In this work, the full rate equations for 17 completely reversible bisubstrate enzyme kinetic mechanisms, with two substrates in the forward and two in the reverse direction, have been presented; among these are rapid equilibrium, steady-state, and mixed steady-state and rapid equilibrium mechanisms. From each rate equation eight product inhibition equations were derived, four for the forward and four for the reverse direction. All the corresponding product inhibition equations were derived in full; thus a total of 17 × 8 = 136 equations, were presented. From these equations a list of product inhibition patterns were constructed and presented in a tabular form, both for the primary plots (intercept effects) and the secondary plots (slope effects).

The purpose of this work is to help investigators in practical work, especially biologists working with enzymes, to choose quickly an appropriate product inhibition pattern for the identification of the kinetic mechanism. The practical application of above product inhibition analysis was illustrated with three examples of yeast alcohol dehydrogenase-catalyzed reactions.     

Deriving the rate equations for product inhibition patterns in bisubstrate enzyme reactions

In this work, the full rate equations for 17 completely reversible bisubstrate enzyme kinetic mechanisms, with two substrates in the forward and two in the reverse direction, have been presented; among these are rapid equilibrium, steady-state, and mixed steady-state and rapid equilibrium mechanisms. From each rate equation eight product inhibition equations were derived, four for the forward and four for the reverse direction. All the corresponding product inhibition equations were derived in full; thus a total of 17 x 8 = 136 equations, were presented. From these equations a list of product inhibition patterns were constructed and presented in a tabular form, both for the primary plots (intercept effects) and the secondary plots (slope effects). The purpose of this work is to help investigators in practical work, especially biologists working with enzymes, to choose quickly an appropriate product inhibition pattern for the identification of the kinetic mechanism. The practical application of above product inhibition analysis was illustrated with three examples of yeast alcohol dehydrogenase-catalyzed reactions.     

Dynamic Disorder in Quasi-Equilibrium Enzymatic Systems

Conformations and catalytic rates of enzymes fluctuate over a wide range of timescales. Despite these fluctuations, there exist some limiting cases in which the enzymatic catalytic rate follows the macroscopic rate equation such as the Michaelis-Menten law. In this paper we investigate the applicability of macroscopic rate laws for fluctuating enzyme systems in which catalytic transitions are slower than ligand binding-dissociation reactions. In this quasi-equilibrium limit, for an arbitrary reaction scheme we show that the catalytic rate has the same dependence on ligand concentrations as obtained from mass-action kinetics even in the presence of slow conformational fluctuations. These results indicate that the timescale of conformational dynamics – no matter how slow – will not affect the enzymatic rate in quasi-equilibrium limit. Our numerical results for two enzyme-catalyzed reaction schemes involving multiple substrates and inhibitors further support our general theory.     

The accuracy of the rapid equilibrium assumption in steady-state enzyme kinetics in the case of a multipath arbitrary enzyme mechanism with a number of equilibrium segments

A quantitative evaluation of the accuracy of the rapid-equilibrium assumption in steady-state enzyme kinetics was obtained for a multipath arbitrary enzyme mechanism with a number of equilibrium segments. Explicit expressions for estimating the contribution of any equilibrium segment to the accuracy of the rapid-equilibrium assumption were obtained. This allowed us to determine the accuracy of the rapid-equilibrium assumption (Δ) in general: 1 + Δ = (1 + Δ₁)(1 + Δ₂)... (1 + Δ_k), where Δ₁, Δ₂,..., Δ_k is the contribution of each individual equilibrium segment. The accuracy depends only on the structure and properties of equilibrium segments, which have been accounted for in the rapid-equilibrium assumption, but it is independent of the number of paths in the mechanism of the enzymatic reaction and on the structure and properties of the remaining part (steady-state) of the kinetic scheme.     

Isoleucyl-tRNA synthetase from baker's yeast. Influence of substrate concentrations on aminoacylation pathways, discrimination between tRNAIle and tRNAVal, and between isoleucine and valine

The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast. For the cognate substrates isoleucine and tRNAIle two Km values each differing by a factor about five were determined; the higher values were observed at concentrations higher than 1 microM, the lower values below 1 microM isoleucine or tRNAIle, respectively. At substrate concentrations below 1 microM also kcat values of the isoleucylation reaction are lowered. With the noncognate substrates valine and tRNAVal such differences could not be detected. The substrate ATP did not show any change of its Km value as far as the reaction was measurable. Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNAIle as the third one; pyrophosphate is the first product to be released, isoleucyl-tRNA the second, and AMP the third one. In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine. From kcat and Km values and from AMP formation rates discrimination factors for discrimination between tRNAIleII and tRNAValI as well as between isoleucine and valine were determined. In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant. The two different Km values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism. Two hypothetical catalytic cycles are postulated.     

The pH-dependence of second-order rate constants of enzyme modification may provide free-reactant pKa values.

1. Reactions of enzymes with site-specific reagents may involve intermediate adsorptive complexes formed by parallel reactions in several protonic states. Accordingly, a profile of the apparent second-order rate constant for the modification reaction (Kobs., the observed rate constant under conditions where the reagent concentration is low enough for the reaction to be first-order in reagent) against pH can, in general, reflect free-reactant-state molecular pKa values only if a quasi-equilibrium condition exists around the reactive protonic state (EHR) of the adsorptive complex. 2. Usually the condition for quasi-equilibrium is expressed in terms of the rate constants around EHR: (formula: see text) i.e. k mod. less than k-2. This often cannot be assessed directly, particularly if it is not possible to determine kmod. 3. It is shown that kmod. must be much less than k-2, however, if kobs. (the pH-independent value of kobs.) less than k+2. 4. Since probable values of k+2 greater than 10(6)M-1.S-1 and since values of kobs. for many modification reactions less than 10(6)M-1.S-1, the equilibrium assumption should be valid, and kinetic study of such reactions should provide reactant-state pKa values. 5. This may not apply to catalyses, because for them the value of kcat./Km may exceed 5 X 10(5)M-1.S-1. 6. The conditions under which the formation of an intermediate complex by parallel pathways may come to quasi-equilibrium are discussed in the Appendix.     

Minimization of intermediate concentrations as a suggested optimality principle for biochemical networks

The multiobjective problem of minimizing all intermediate concentrations is solved for a model of glycolysis, the pentose monophosphate shunt and the glutathione system in human erythrocytes. It turns out that one solution out of four obtained corresponds qualitatively to the real system. Furthermore, it is shown that for any reaction system, the mentioned optimality principle implies distinct time hierarchy in that some reactions are infinitely fast and subsist in quasi-equilibrium. Finally, the relationships to the standard method of deriving enzymatic rate laws are discussed.