Evolutionary optimization of the catalytic efficiency of enzymes.

1. The rate equation for a generalized Michaelian type of enzymic reaction mechanism has been analyzed in order to establish how the mechanism should be kinetically designed in order to optimize the catalytic efficiency of the enzyme for a given average magnitude of true and apparent first-order rate constants in the mechanism at given concentrations of enzyme, substrate and product. 2. As long as on-velocity constants for substrate and product binding to the enzyme have not reached the limiting value for a diffusion-controlled association process, the optimal state of enzyme operation will be characterized by forward (true and apparent) first-order rate constants of equal magnitude and reverse rate constants of equal magnitude. The drop in free energy driving the catalysed reaction will occur to an equal extent for each reaction step in the mechanism. All internal equilibrium constants will be of equal magnitude and reflect only the closeness of the catalysed reaction to equilibrium conditions. 3. When magnitudes of on-velocity constants for substrate and product binding have reached their upper limits, the optimal kinetic design of the reaction mechanism becomes more complex and has to be established by numerical methods. Numerical solutions, calculated for triosephosphate isomerase, indicate that this particular enzyme may or may not be considered to exhibit close to maximal efficiency, depending on what value is assigned to the upper limit for a ligand association rate constant. 4. Arguments are presented to show that no useful information on the evolutionary optimization of the catalytic efficiency of enzymes can be obtained by previously taken approaches that are based on the application of linear free-energy relationships for rate and equilibrium constants in the reaction mechanism.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

A novel kinetic mechanism explaining the non-hyperbolic behavior of metal activated enzymes. Case of choline kinase from rat liver

A novel kinetic mechanism that explains the non-hyperbolic kinetics of many metal- or effector-activated enzymes is proposed as an alternative to the allosteric, hysteresis and mnemonical models. In this mechanism, the non-Michaelian behavior is generated by a reversible binding of an essential metal cation or other effector to a single site, but to at least two different enzyme forms in steady state. The model is described by a higher degree rate equation since the metal binding to more than one enzyme form generates at least two steady-state catalytic pathways of different efficiencies in addition to the recycling of the metal-enzyme species in the kinetic sequence. The proposed mechanism also explains the transition from non-hyperbolic kinetics to a Michaelian rate law, as well as the dual activation and inhibition of enzymes by the metal cation or effector, according to its concentration. This kinetic behavior is generated by the participation of the metal or effector in fast and slow competing catalytic sequences or by the competitions produced by binding as a common reactant for both the forward and reverse reactions. The model can also explain some peculiar inhibition patterns observed for some transferases. This kinetic mechanism can be tested by an experimental protocol that includes the metal cation or effector as a controlled variable reactant. The model and its complete rate equation explains the non-Michaelian behavior of choline kinase. At low ligand concentrations, an effectively ordered terreactant sequential mechanism operates (Infant. & Kinsella, 1976). The steady-state addition of choline to free enzyme is followed by the rapid-equilibrium binding of MgATp^2? and the steady-state addition of Mg²⁺ last in the sequence. Initial velocity and product inhibition studies in the non-hyperbolic kinetic region, were consistent with a partially ordered release of reactants in which phosphocholine was the first product to dissociate from the central complex. The release sequence of the other two reactants was dependent on the prevailing Mg²⁺ concentration. At low Mg²⁺ levels, i.e. below 2·0 mM the metal cation is predominantly released after phosphocholine whereas MgADP^? is the last product to dissociate under rapid-equilibrium conditions. At higher levels of the metal cation, MgADP^? is predominantly released after phosphocholine leaving the Mg-enzyme complex from which Mg²⁺ may dissociate. However, a substantial fraction of the Mg-enzyme form is recycled in an alternate catalytic sequence in which the rapid-equilibrium binding MgATP^2? to the Mg-enzyme complex is followed by the steady-state addition of choline. This pathway can also be initiated by the binding of Mg²⁺ to free enzyme. A third and unique sequence, which operates at low Mg²⁺ concentrations, includes the participation of MgADP^? as an activator via a partial reversal of one of the product release sequences. In this pathway, the rapidequilibrium binding of MgADP^? to free enzyme is followed by the addition of Mg²⁺ to the resulting transitory complex. Subsequent dissociation of MgADP^? leaves the Mg-enzyme form, which is then channeled to product formation by the consecutive additions of MgATP^2? and choline.     

Theory of the kinetics of reactions catalyzed by enzymes attached to membranes

A theoretical treatment has been worked out for the kinetics of solid-supported enzyme systems, with diffusive and electrostatic effects taken into account. A utilization factor, defined as the ratio of the actual reaction rate to the rate of substrate consumption in the outer solution, is defined, and equations to evaluate the utilization factor are given for five kinetic conditions: (a) Michaelis-Menten behavior, (b) substrate inhibition, (c) product inhibition (competitive), (d) product inhibition (noncompetitive), and (e) product inhibition (anticompetitive). When the solid-supported enzymes obey a Michaelis-Menten relationship, an equation for the apparent Michaelis constant is given and a criterion for insignificant diffusion effects is shown. A substrate-inhibited enzyme reaction may display multiple steady-state behavior, and a criterion for uniqueness is presented. In the case of product-inhibited enzyme reactions, the utilization factor is always less than that which corresponds to a Michaelis-Menten relationship. Equations to evaluate the apparent Michaelis and inhibition constants are given.     

Mechanistic studies of Escherichia coli adenosine-5'-phosphosulfate kinase

Adenosine-5'-phosphosulfate kinase (APS kinase) catalyzes the formation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the major form of activated sulfate in biological systems. The enzyme from Escherichia coli has complex kinetic behavior, including substrate inhibition by APS and formation of a phosphorylated enzyme (E-P) as a reaction intermediate. The presence of a phosphorylated enzyme potentially enables the steady-state kinetic mechanism to change from sequential to ping-pong as the APS concentration decreases. Kinetic and equilibrium binding measurements have been used to evaluate the proposed mechanism. Equilibrium binding studies show that APS, PAPS, ADP, and the ATP analog AMPPNP each bind at a single site per subunit; thus, substrates can bind in either order. When ATPgammaS replaces ATP as substrate the V(max) is reduced 535-fold, the kinetic mechanism is sequential at each APS concentration, and substrate inhibition is not observed. The results indicate that substrate inhibition arises from a kinetic phenomenon in which product formation from ATP binding to the E. APS complex is much slower than paths in which product formation results from APS binding either to the E. ATP complex or to E-P. APS kinase requires divalent cations such as Mg(2+) or Mn(2+) for activity. APS kinase binds one Mn(2+) ion per subunit in the absence of substrates, consistent with the requirement for a divalent cation in the phosphorylation of APS by E-P. The affinity for Mn(2+) increases 23-fold when the enzyme is phosphorylated. Two Mn(2+) ions bind per subunit when both APS and the ATP analog AMPPNP are present, indicating a potential dual metal ion catalytic mechanism.     

A computer program for enzyme kinetics that combines model discrimination, parameter refinement and sequential experimental design.

A method of model discrimination and parameter estimation in enzyme kinetics is proposed. The experimental design and analysis of the model are carried out simultaneously and the stopping rule for experimentation is deduced by the experimenter when the probabilities a posteriori indicate that one model is clearly superior to the rest. A FORTRAN77 program specifically developed for joint designs is given. The method is very powerful, as indicated by its usefulness in the discrimination between models. For example, it has been successfully applied to three cases of enzyme kinetics (a single-substrate Michaelian reaction with product inhibition, a single-substrate complex reaction and a two-substrate reaction). By using this method the most probable model and the estimates of the parameters can be obtained in one experimental session. The FORTRAN77 program is deposited as Supplementary Publication SUP 50134 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5.     

Structured kinetic model to represent the utilization of multiple substrates in complex media during rifamycin B fermentation

Industrial fermentations typically use media that are balanced with multiple substitutable substrates including complex carbon and nitrogen source. Yet, much of the modeling effort to date has mainly focused on defined media. Here, we present a structured model that accounts for growth and product formation kinetics of rifamycin B fermentation in a multi-substrate complex medium. The phenomenological model considers the organism to be an optimal strategist with an in-built mechanism that regulates the sequential and simultaneous uptake of the substrate combinations. This regulatory process is modeled by assuming that the uptake of a substrate depends on the level of a key enzyme or a set of enzymes, which may be inducible. Further, the fraction of flux through a given metabolic branch is estimated using a simple multi-variable constrained optimization. The model has the typical form of Monod equation with terms incorporating multiple limiting substrates and substrate inhibition. Several batch runs were set up with varying initial substrate concentrations to estimate the kinetic parameters for the rifamycin overproducer strain Amycolatopsis mediterranei S699. Glucose and ammonium sulfate (AMS) demonstrated significant substrate inhibition toward growth as well as product formation. The model correctly predicts the experimentally observed regulated simultaneous uptake of the substitutable substrate combinations under different fermentation conditions. The modeling results may have applications in the optimization and control of rifamycin B fermentation while the modeling strategy presented here would be applicable to other industrially important fermentations.     

Comparison of the enzymatic behavior of high molecular weight and free lysyl-tRNA synthetase from rat liver: kinetic analysis of lysylation of tRNA

Lysyl-tRNA synthetase occurs in the high molecular weight form in rat liver. The high molecular weight lysyl-tRNA synthetase has been previously demonstrated to exist as multienzyme complexes of aminoacyl-tRNA synthetases. The multienzyme complexes can be dissociated by hydrophobic interaction chromatography and yield fully active, free lysyl-tRNA synthetase. The free form is found to be twice as active as the complexed form in lysylation. Bisubstrate and product inhibition kinetics of lysylation are systematically carried out for highly purified free lysyl-tRNA synthetase and the 18 S synthetase complex. Surprisingly, the two enzyme forms exhibit distinctly different kinetic patterns in bisubstrate and product inhibition kinetics under identical conditions. The 18 S synthetase complex shows kinetic patterns consistent with an ordered bi uni uni bi ping pong mechanism, while the results of free lysyl-tRNA synthetase do not. We conclude that structural organization of lysyl-tRNA synthetase beyond quaternary structure of proteins may alter the enzyme behavior.     

The kinetic effect of product instability in a Michaelis-Menten mechanism with competitive inhibition

In most kinetic studies it is assumed that both the reactant and the products are stable. However, under certain conditions spontaneous decomposition or deterioration caused by one of the participating species occurs. Studies, in which a species (the free enzyme, the enzyme-substrate complex, an inhibitor or the product of the reaction) is unstable, have appeared in the literature. However, to our knowledge, the enzymatic systems, in which a competitive inhibition and a decomposition or transformation of the products take place simultaneously, have not been studied so far. In this paper, we present a kinetic analysis of an enzyme reaction that follows a Michaelis-Menten mechanism, in which the free enzyme suffers a competitive inhibition simultaneously with the decomposition of the immediate product. In this study, we have linearised the differential equations that describe the kinetics of the process. Under the assumption of limiting concentration of enzyme, we have obtained and tested the explicit equation describing the time dependence of the product concentration using numerical calculus. With this equation and the experimental progress curve of the product, we constructed an easy procedure for the evaluation of the principal kinetic parameters of the process.     

Acetic acid formation in Escherichia coli fermentation

Theoretical analysis of cellulase product inhibition (by cellobiose and glucose) has been performed in terms of the mathematical model for enzymatic cellulose hydrolysis. The analysis showed that even in those cases when consideration of multienzyme cellulase system as one enzyme (cellulase) or two enzymes (cellulase and beta-glucosidase) is valid, double-reciprocal plots, usually used in a product inhibition study, may be nonlinear, and different inhibition patterns (noncompetitive, competitive, or mixed type) may be observed. Inhibition pattern depends on the cellulase binding constant, enzyme concentration, maximum adsorption of the enzyme (cellulose surface area accessible to the enzyme), the range in which substrate concentration is varied, and beta-glucosidase activity. A limitation of cellulase adsorption by cellulose surface area that may occur at high enzyme/substrate ratio is the main reason for nonlinearity of double-reciprocal plots. Also, the results of calculations showed that material balance by substrate, which is usually neglected by researchers studying cellulase product inhibition, must be taken into account in kinetic analysis even in those cases when the enzyme concentration is rather low. (c) 1992 John Wiley & Sons, Inc.     

The analysis of enzyme progress curves by numerical differentiation, including competitive product inhibition and enzyme reactivation

A new method for analyzing steady-state enzyme kinetic data is presented. The technique, which is based on the numerical differentiation of the complete reaction curve, has several advantages over initial velocity and integrated Michaelis-Menten equation methods. The differentiated data are fit to the differential equation describing the appropriate kinetic scheme. This approach is particularly valuable in cases of strong competitive product inhibition and of changing concentrations of active enzyme. The method assumes a reversible reaction and is applicable to a very wide variety of steady-state kinetic schemes. A particular advantage of this approach over integrated methods is that it is independent of [S0] and hence of errors in [S0]. The combination of complete progress curve and computer analysis makes this approach very efficient with respect to both time and materials. Running on an IBM PC XT or equivalent microcomputer with an 8087 coprocessor, the analyses are very fast, the complete process usually being complete in a minute or two. The utility of the technique is demonstrated by application to both simulated and real data. We show that the differentiation of the progress curve for the ribonuclease-catalyzed hydrolysis of 2',3'-cyclic cytidine monophosphate reveals strong product inhibition by 3'-CMP, and this product inhibition accounts for the large discrepancies reported in the literature for the value of Km for this substrate. The method was also applied to determine the rate of reactivation of beta-lactamase which had been reversibly inactivated by cloxacillin. Since large numbers of data points are required for the numerical differentiation the method has become practical only with the advent of computer-acquired data systems.     

A slow-tight binding inhibitor of dopamine beta-monooxygenase: a transition state analogue for the product release step

The steady-state kinetic data show that 3-hydroxy-4-phenylthiazole-2(3H)-thione (3H4PTT) is a potent tight-binding inhibitor for dopamine beta-monooxygenase (DbetaM) with a dissociation constant of 0.9 nM. Ackermann-Potter plots of the enzyme dependence of the inhibition revealed that the stoichiometry of the enzyme inhibition by 3H4PTT is 1:1. Pre-steady-state progress curves at varying inhibitor with fixed reductant and enzyme concentrations clearly show the slow binding behavior of the inhibitor. The observed kinetic behavior is consistent with the apparent direct formation of the tightly bound E x I* complex. The k(on) and k(off) for 3H4PTT which were determined under pre-steady-state conditions at variable inhibitor concentrations were found to be (1.85 +/- 0.07) x 10(6) M(-1) s(-1) and (1.9 +/- 0.6) x 10(-3) s(-1), respectively. The dissociation constant calculated from these rates was similar to that determined under steady-state conditions, confirming that 3H4PTT is a kinetically well-behaved inhibitor. The steady-state as well as pre-steady-state kinetic studies at variable DMPD concentrations show that the inhibition is competitive with respect to the reductant, demonstrating the exclusive interaction of 3H4PTT with the oxidized form of the enzyme. The kinetic behavior and the structural properties of 3H4PTT are consistent with the proposal that the E x 3H4PTT complex may mimic the transition state for the product (protonated) release step of the enzyme. Therefore, 3H4PTT could be used as a convenient probe to examine the properties of the E x P complex of the DbetaM reaction and also as an active site titrant for the oxidized enzyme.     

Purine nucleoside phosphorylase of rabbit liver. Mechanism of catalysis.

Initial velocity studies and product inhibition patterns for purine nucleoside phosphorylase from rabbit liver were examined in order to determine the predominant catalytic mechanism for the synthetic (forward) and phosphorolytic (reverse) reactions of the enzyme. Initial velocity studies in the absence of products gave intersecting or converging linear double reciprocal plots of the kinetic data for both the synthetic and phosphorolytic reactions of the enzyme. The observed kinetic pattern was consistent with a sequential mechanism, requiring that both substrates add to the enzyme before products may be released. The product inhibition patterns showed mutual competitive inhibition between guanine and guanosine as variable substrates and inhibitors. Ribose 1-phosphate and inorganic orthophosphate were also mutually competitive toward each other. Other combinations of substrates and products gave noncompetitive inhibition. Apparent inhibition constants calculated for guanine as competitive inhibitor and for ribose 1-phosphate as noncompetitive inhibitor of the enzyme, with guanosine as variable substrate, did not vary significantly with increasing concentrations of inorganic orthophosphate as fixed substrate. These results suggest that the mechanism was order and that substrates add to the enzyme in an obligatory order. Dead end inhibition studies carried out in the presence of the products guanine and ribose 1-phosphate, respectively, showed that the kinetically significant abortive ternary complexes of enzyme-guanine-inorganic orthophosphate (EQB) and enzyme-guanose-ribose 1-phosphate (EAP) are formed. The results of dead end inhibition studies are consistent with an obligatory order of substrate addition to the enzyme. The nucleoside or purine is probably the first substrate to form a binary complex with the enzyme, and with which inorganic orthophosphate or ribose 1-phosphate may interact as secondary substrates. The evidences presented in this investigation support an Ordered Theorell-Chance mechanism for the enzyme.     

Polyphosphate kinase from M. tuberculosis: an interconnect between the genetic and biochemical role

The enzyme Polyphosphate Kinase (PPK) catalyses the reversible transfer of the terminal γ-Pi of ATP to form a long chain Polyphosphate (PolyP). Using an IPTG inducible mycobacterial vector, the vulnerability of this gene has been evaluated by antisense knockdown experiments in M. tuberculosis. Expression profiling studies point to the fact that down regulation of PPK caused cidality during the late phase in contrast to its bacteriostatic mode immediately following antisense expression. PPK thus seems to be a suitable anti-tubercular drug target. The enzyme which is a tetramer has been cloned in E. coli and purified to homogeneity. An enzyme assay suitable for High Throughput Screening was optimized by using the statistical Taguchi protocol and the kinetic parameters determined. The enzyme displayed a strong product inhibition by ADP. In order to accurately estimate the product inhibition, progress curve analysis of the enzyme reaction was monitored. The kinetic equation describing the progress curve was suitably modified by taking into account the product inhibition. The reversible nature of the enzyme indicated a possibility of a two way ATP↔ADP switch operating in the bacteria as a response to its growth requirement.     

Removal from the Membrane Affects the Interaction of Rat Osseous Plate Ecto-Nucleosidetriphosphate Diphosphohydrolase-1 with Substrates and Ions

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M (r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always <2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.     

Regulation of human lymphocyte S-adenosylmethionine synthetase by product inhibition

A steady-state kinetic analysis of human S-adenosylmethionine synthetase indicates that the reaction is Bi Ter with ordered addition of ATP and L-methionine and release of S-adenosylmethionine as the first product. Pyrophosphate and phosphate are then released randomly. I-Parabolic inhibition by phosphate with respect to ATP indicates that this product must bind to more than one site. A model in which phosphate binds to the pyrophosphate site gives a rate equation that is consistent with the kinetic data. Values have been determined for those constants in the equation that are large enough to evaluate, and the in vitro kinetic behavior of S-adenosylmethionine synthetase can be predicted at substrate and product concentrations that are expected intracellularly. Inhibition by combinations of products, especially pyrophosphate and phosphate, is synergistic. Of particular interest is the ability of pyrophosphate and phosphate to increase the sensitivity of the enzyme to inhibition by S-adenosylmethionine. This phenomenon may play a role in regulating steady-state cellular concentrations of S-adenosylmethionine.     

Adenosine-5'-phosphosulfate kinase from Escherichia coli K12. Purification, characterization, and identification of a phosphorylated enzyme intermediate

Adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase), the second enzyme in the pathway of sulfate activation, has been purified (approximately 300-fold) to homogeneity from an Escherichia coli K12 strain, which overproduces the enzyme activity (approximately 100-fold). The purified enzyme has a specific activity of 153 mumol of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) formed/min/mg of protein at 25 degrees C. The enzyme is remarkably efficient with a Vmax/Km(APS) of greater than 10(8) M-1 s-1, indicating that at physiologically low substrate concentrations the reaction is essentially diffusion limited. Upon incubation with MgATP a phosphorylated enzyme is formed; the isolated phosphorylated enzyme can transfer its phosphoryl group to adenosine 5'-phosphosulfate (APS) to form PAPS or to ADP to form ATP. The phosphorylated enzyme exists as a dimer of identical 21-kilodalton subunits, while the dephosphorylated form primarily exists as a tetramer. Divalent cations are required for activity with Mg(II), Mn(II), Co(II), and Cd(II) activating. Studies of the divalent metal-dependent stereoselectivity for the alpha- and beta-phosphorothioate derivatives of ATP indicate metal coordination to at least the alpha-phosphoryl group of the nucleotide. Steady state kinetic studies of the reverse reaction indicate a sequential mechanism, with a rapid equilibrium ordered binding of MgADP before PAPS. In the forward direction APS is a potent substrate inhibitor, competitive with ATP, complicating kinetic studies. The primary kinetic mechanism in the forward direction is sequential. Product inhibition studies at high concentrations of APS suggest an ordered kinetic mechanism with MgATP binding before APS. At submicromolar concentrations of APS, product inhibition by both MgADP and PAPS is more complex and is not consistent with a solely ordered sequential mechanism. The formation of a phosphorylated enzyme capable of transferring its phosphoryl group to APS or to MgADP suggests that a ping-pong pathway in which the rate of MgADP dissociation is comparable to the rate of APS binding might contribute at very low concentrations of APS. The substrate inhibition by APS is consistent with APS binding to the enzyme, to form a dead-end E.APS complex.     

Chorismate lyase: kinetics and engineering for stability

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.     

Complete kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae

The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated to be 11 +/- 2 mM as measured using Mg2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg2+ and alpha-ketoadipate. Curvature is observed in the double-reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases, the inhibition changes to uncompetitive, consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO2, alpha-ketoadipate, and NADH. Isocitrate is a slow substrate with a rate (V/E(t)) 216-fold slower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg2+ bind to the enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The Keq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.     

Kinetic studies on beta-site amyloid precursor protein-cleaving enzyme (BACE). Confirmation of an iso mechanism

The steady-state kinetic mechanism of beta-amyloid precursor protein-cleaving enzyme (BACE)-catalyzed proteolytic cleavage was evaluated using product and statine- (Stat(V)) or hydroxyethylene-containing (OM99-2) peptide inhibition data, solvent kinetic isotope effects, and proton NMR spectroscopy. The noncompetitive inhibition pattern observed for both cleavage products, together with the independence of Stat(V) inhibition on substrate concentration, suggests a uni-bi-iso kinetic mechanism. According to this mechanism, the enzyme undergoes multiple conformation changes during the catalytic cycle. If any of these steps are rate-limiting to turnover, an enzyme form preceding the rate-limiting conformational change should accumulate. An insignificant solvent kinetic isotope effect (SKIE) on k(cat)/K(m), a large inverse solvent kinetic isotope effect on k(cat), and the absence of any SKIE on the inhibition onset by Stat(V) during catalysis together indicate that the rate-limiting iso-step occurs after formation of a tetrahedral intermediate. A moderately short and strong hydrogen bond (at delta 13.0 ppm and phi of 0.6) has been observed by NMR spectroscopy in the enzyme-hydroxyethylene peptide (OM99-2) complex that presumably mimics the tetrahedral intermediate of catalysis. Collapse of this intermediate, involving multiple steps and interconversion of enzyme forms, has been suggested to impose a rate limitation, which is manifested in a significant SKIE on k(cat). Multiple enzyme forms and their distribution during catalysis were evaluated by measuring the SKIE on the noncompetitive (mixed) inhibition constants for the C-terminal reaction product. Large, normal SKIE values were observed for these inhibition constants, suggesting that both kinetic and thermodynamic components contribute to the K(ii) and K(is) expressions, as has been suggested for other iso-mechanism featuring enzymes. We propose that a conformational change related to the reprotonation of aspartates during or after the bond-breaking event is the rate-limiting segment in the catalytic reaction of beta-amyloid precursor protein-cleaving enzyme, and ligands binding to other than the ground-state forms of the enzyme might provide inhibitors of greater pharmacological relevance.