Substrate binding to chloramphenicol acetyltransferase: evidence for negative cooperativity from equilibrium and kinetic constants for binary and ternary complexes

Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol by a ternary complex mechanism with a rapid equilibrium and essentially random order of addition of substrates. Such a kinetic mechanism for a two-substrate reaction provides an opportunity to compare the affinity of enzyme for each substrate in the binary complexes (1/Kd) with corresponding values (1/Km) for affinities in the ternary complex where any effect of the other substrate should be manifest. The pursuit of such information for CAT involved the use of four independent methods to determine the dissociation constant (Kd) for chloramphenicol in the binary complex, techniques which included stopped-flow measurements of on and off rates, and a novel fluorometric titration method. The binary complex dissociation constant (Kd) for acetyl-CoA was measured by fluorescence enhancement and steady-state kinetic analysis. The ternary complex dissociation constant (Km) for each substrate (in the presence of the other) was determined by kinetic and fluorometric methods, using CoA or ethyl-CoA to form nonproductive ternary complexes. The results demonstrate an unequivocal decrease in affinity of CAT for each of its substrates on progression from the binary to the ternary complex, a phenomenon most economically described as negative cooperativity. The binary complex dissociation constants (Kd) for chloramphenicol and acetyl-CoA are 4 microM and 30 microM whereas the corresponding dissociation constants in the ternary complex (Km) are 12 microM and 90 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic mechanism of the hydroxypyruvate-lactate dehydrogenase-NADH system.

Chicken liver lactate dehydrogenase L-lactate : NAD+ oxidoreductase, EC1.1.1.27) reversibly catalyses the conversion of hydroxypyruvate to L-glycerate. The variation of the initial reaction rate with the substrate or coenzyme (NADH) concentration together with the inhibition caused by the reaction products and excess substrates, reveal that the kinetic mechanism of the reaction, with hydroxypyruvate as substrate, is of the rapid-equilibrium, ordered-ternary-complex type; NADH is the first substrate in the reaction sequence. Rate equations have been developed for the hydroxypyruvate.E.NADH system without inhibitors, with excess substrates, and with reaction products. Comparison of the rate equations obtained with those calculated theoretically from an ordered-ternary-complex mechanism reveals the existence of E.NAD.NADH,E.NAD-hydroxypyruvate and E.hydroxypyruvate complexes.     

A steady-state random-order mechanism for the oxidative deamination of norvaline by glutamate dehydrogenase.

The kinetic mechanism of glutamate dehydrogenase with the monocarboxylic substrate norvaline was examined by using initial-rate steady-state kinetics and inhibition kinetics. To a first approximation the reaction mechanism can be described as a rapid-equilibrium random-order one. Binding synergism between the monocarboxylic substrate and coenzyme is not observed. Dissociation constants for NAD+ and 2-oxoglutarate calculated from the kinetic data assuming a rapid-equilibrium random-order model are in good agreement with independently obtained estimates. Lineweaver-Burk plots with varied norvaline concentration are not strictly linear, and it is concluded that a steady-state random-order model more accurately reflects the observed kinetics with norvaline as substrate.     

Vitamin K-dependent carboxylase from calf liver: studies on the steady-state kinetic mechanism

The steady-state kinetic mechanism of vitamin K-dependent carboxylase from calf liver has been investigated by initial-velocity measurements with varying concentrations of two carboxylase substrates and constant, nonsaturating concentrations of the other two substrates. With all combinations of the varied substrates tested linear kinetics were obtained with lines intersecting on the left side of the 1/v axis in double-reciprocal plots. Thus the carboxylase has a sequential reaction mechanism which includes the quinternary complex of the enzyme with its four substrates. A mechanism with the ordered steady-state addition of all substrates to the enzyme accords well with the results. A totally random mechanism was excluded but the alternative possibility remained that part of the substrates are added in a rapid-equilibrium random reaction. Experiments with saturating constant concentrations of sodium bicarbonate and varying concentrations of the other substrates suggest that bicarbonate (CO2) is either the first or, more probably, the last substrate bound to the enzyme.     

Uridine phosphorylase from Escherichia coli B.: kinetic studies on the mechanism of catalysis

Using a highly purified enzyme preparation of uridine phosphorylase from Escherichia coli B, we have performed detailed kinetic studies which include initial-velocity and product-inhibition experiments in the forward and reverse directions of the reaction. These studies indicate a rapid-equilibrium random mechanism for this enzyme with the formation of an enzyme . uracil phosphate abortive complex. Lack of formation of the enzyme . uridine . ribose-1-phosphate abortive complex suggests that the ribosyl moiety of the two ligands compete for the same binding site. The random mechanism is different from the ordered addition of substrates found for uridine phosphorylase from other sources. All the kinetic constants in the forward and reverse directions and the Keq of reaction for E. coli uridine phosphorylase are reported herein.     

The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker''s yeast

1. The effect of independent variation of both acetyl-CoA and acetoacetyl-CoA on the initial velocity at pH8.0 and pH8.9 gives results compatible with a sequential mechanism involving a modified enzyme tentatively identified as an acetyl-enzyme, resulting from the reaction with acetyl-CoA in the first step of a Ping Pong (Cleland, 1963a) reaction. 2. Acetoacetyl-CoA gives marked substrate inhibition that is competitive with acetyl-CoA. This suggests formation of a dead-end complex with the unacetylated enzyme and is in accord with the inhibition pattern given by 3-oxohexanoyl-CoA, an inactive analogue of acetoacetyl-CoA. 3. The inhibition pattern given by products of the reaction is compatible with the above mechanism. CoA gives mixed inhibition with respect to both substrates, whereas dl-3-hydroxy-3-methylglutaryl-CoA competes with acetyl-CoA but gives uncompetitive inhibition with respect to acetoacetyl-CoA. 4. 3-Hydroxy-3-methylglutaryl-CoA analogues lacking the 3-hydroxyl group are found to compete, like 3-hydroxy-3-methylglutaryl-CoA, with acetyl-CoA but have K(i) values ninefold higher, indicating the importance of the 3-hydroxyl group in the interaction. 5. A comparison of inhibition by CoA and desulpho-CoA at pH8.0 and pH8.9 shows that at the higher pH value a kinetically significant reversal of the formation of acetyl-enzyme can occur. 6. Acetyl-CoA homologues do not act as substrates and compete only with acetyl-CoA. A study of the variation of K(i) with acyl-chain length suggests the presence near the active centre of a hydrophobic region. 7. These results are discussed in terms of a kinetic mechanism in which there is only one CoA-binding site the specificity of which is altered by acetylation of the enzyme. 8. The rate of 3-hydroxy-3-methylglutaryl-CoA synthesis in yeast is calculated from the kinetic constants determined for purified 3-hydroxy-3-methylglutaryl-CoA synthase and from estimates of the physiological substrate concentrations. The rate of synthesis of 12nmol of 3-hydroxy-3-methylglutaryl-CoA/min per g wet wt. of yeast is still greater than the rate of utilization in spite of the extremely low (calculated) acetoacetyl-CoA concentration (1.8nm).     

Recombinant human glucose-6-phosphate dehydrogenase. Evidence for a rapid-equilibrium random-order mechanism.

Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver-Burk plots as expected for a ternary-complex mechanism. Patterns of product and dead-end inhibition indicated that the enzyme can bind NADP+ and Glc6P separately to form binary complexes, suggesting a random-order mechanism. The Kd value for the binding of NADP+ measured by titration of protein fluorescence is 8.0 microm, close to the value of 6.8 microm calculated from the kinetic data on the assumption of a rapid-equilibrium random-order mechanism. Strong evidence for this mechanism and against either of the compulsory-order possibilities is provided by repeating the kinetic analysis with each of the natural substrates replaced in turn by structural analogues. A full kinetic analysis was carried out with deaminoNADP+ and with deoxyglucose 6-phosphate as the alternative substrates. In each case the calculated dissociation constant upon switching a substrate in a random-order mechanism (e.g. that for NADP+ upon changing the sugar phosphate) was indeed constant within experimental error as expected. The calculated rate constants for binding of the leading substrate in a compulsory-order mechanism, however, did not remain constant when the putative second substrate was changed. Previous workers, using enzyme from pooled blood, have variously proposed either compulsory-order or random-order mechanisms. Our study appears to provide unambiguous evidence for the latter pattern of substrate binding.     

Kinetic mechanism of the type II calmodulin-dependent protein kinase: studies of the forward and reverse reactions and observation of apparent rapid-equilibrium ordered binding

The kinetic reaction mechanism of the type II calmodulin-dependent protein kinase was studied by using its constitutively active kinase domain. Lacking regulatory features, the catalytic domain simplified data collection, analysis, and interpretation. To further facilitate this study, a synthetic peptide was used as the kinase substrate. Initial velocity measurements of the forward reaction were consistent with a sequential mechanism. The patterns of product and dead-end inhibition studies best fit an ordered Bi Bi kinetic mechanism with ATP binding first to the enzyme, followed by binding of the peptide substrate. Initial-rate patterns of the reverse reaction of the kinase suggested a rapid-equilibrium mechanism with obligatory ordered binding of ADP prior to the phosphopeptide substrate; however, this apparent rapid-equilibrium ordered mechanism was contrary to the observed inhibition by the phosphopeptide which is not supposed to bind to the kinase in the absence of ADP. Inspection of product inhibition patterns of the phosphopeptide with both ATP and peptide revealed that an ordered Bi Bi mechanism can show initial-rate patterns of a rapid-equilibrium ordered system when a Michaelis constant for phosphopeptide, Kip, is large relative to the concentration of phosphopeptide used. Thus, the results of this study show an ordered Bi Bi mechanism with nucleotide binding first in both directions of the kinase reaction. All the kinetic constants in the forward and reverse directions and the Keq of the kinase reaction are reported herein. To provide theoretical bases and diagnostic aid for mechanisms that can give rise to typical rapid-equilibrium ordered kinetic patterns, a discussion on various sequential cases is presented in the Appendix.     

Kinetic properties of Cellulomonas sp. purine nucleoside phosphorylase with typical and non-typical substrates: implications for the reaction mechanism

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m7Guo), with either nucleoside or phosphate (Pd) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of Pi, a substrate, and also a reaction modifier that helps to release a tightly bound purine base.     

Fatty acid synthetase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from pigeon liver.

The kinetic mechanism of pigeon liver fatty acid synthetase action has been studied using steady state kinetic analysis. Initial velocity studies are consistent with an earlier suggestion that the enzyme catalyzes this reaction by a seven-site ping-pong mechanism. Although the range of substrate concentrations that could be used was limited by several factors, the initial velocity patterns showing the relationship between the substrates acetyl coenzyme CoA, malonyl-CoA, and NADPH appear to be a series of parallel lines, regardless of which substrate is varied at fixed levels of a second substrate. However, two of the substrates, acetyl-CoA and malonly-CoA, apparently exhibit a competitive substrate inhibition with respect to each other, but NADPH shows no inhibition of any kind. Product inhibition patterns suggest that free CoA is competitive versus acetyl-CoA and malonyl-CoA and is uncompetitive versus NADPH, and that NADP+ is competitive versus NADPH and uncompetitive versus acetyl-CoA or malonyl-CoA. These results are consistent with a seven-site ping-pong mechanism with intermediates covalently bound to 4'-phosphopantetheine (part of acyl carrier protein). Double competitive substrate inhibition by acetyl-CoA and malonyl-CoA is consistent with the rate equation derived for the over-all mechanism. The kinetic mechanism developed from these results is capable of explaining the formation of fatty acids from malonyl-CoA and NADPH alone (Katiyar, S. S., Briedis, A. V., and Porter, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420) and also the formation of triacetic acid lactone from either malonyl-CoA alone or acetyl-CoA plus malonyl-CoA.     

Reaction mechanism, specificity and pH-dependence of peptide synthesis catalyzed by the metalloproteinase thermolysin

The initial rates of peptide bond formation catalyzed by the metalloproteinase thermolysin were determined. The dependence of the formation rates on the concentration of the carboxyl donor and the acceptor can be explained by a rapid-equilibrium random bireactant mechanism, in which the binding of one substrate has a positive influence on the binding of the other (synergism). The specificity of the enzyme for the donor and acceptor in the condensation reaction was further investigated by determining the apparent kinetic parameters kcat and Km for various substrates. The pH-dependence of the initial rates of synthesis was found to be identical to the pH-dependence of the hydrolytic action of the enzyme. The rates are also shown to be independent of the pKa of the amino group of the acceptor, indicating that deprotonation of the attacking nucleophile in the synthetic reaction is not rate-limiting.     

Bacterial formate dehydrogenase. Substrate specificity and kinetic mechanism of S-formyl glutathione oxidation

The substrate specificity of NAD-dependent formate dehydrogenase from the methylotrophic bacterium Achromobacter parvulus T1 was studied. The kinetic mechanism of S-formyl glutathione oxidation was determined. The initial velocity studies and inhibition analysis were carried out. It was shown that the kinetic mechanism for the enzyme with S-formyl glutathione as a substrate is similar to that with formate and is rapid-equilibrium random. Using independent methods, it was found that formate dehydrogenase forms a binary complex with S-formyl glutathione (Kd = 2.5 mM).     

Kinetic mechanism of antiports catalyzed by reconstituted ornithine/citrulline carrier from rat liver mitochondria

The transport mechanism of the reconstituted ornithine/citrulline carrier purified from rat liver mitochondria was investigated kinetically. A complete set of half-saturation constants (K(m)) was established for ornithine, citrulline and H(+) on both the external and internal side of the liposomal membrane. The internal affinity for ornithine was much lower than that determined on the external surface. The exclusive presence of a single transport affinity for ornithine on each side of the membrane indicated a unidirectional insertion of the ornithine/citrulline carrier into liposomes, probably right-side-out with respect to mitochondria. Two-reactant initial velocity studies of the homologous (ornithine/ornithine) and heterologous (ornithine/citrulline) exchange reactions resulted in a kinetic pattern which is characteristic of a simultaneous antiport mechanism. This type of mechanism implies that the carrier forms a ternary complex with the substrates before the transport reaction occurs. A quantitative analysis of substrate interaction revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrates.     

Purification and properties of acetyl-CoA:L-glutamate N-acetyltransferase from human liver.

Acetyl-CoA:L-glutamate N-acetyltransferase (amino acid acetyltransferase, EC 2.3.1.1) was isolated from human liver mitochondria by precipitation with (NH4)2SO4 and chromatography on hydroxyapatite, DEAE-cellulose and Sephacryl 300. This gave a 360-fold purification. The molecular weight was estimated to be approx. 190 000. The kinetic properties in the absence of arginine are compatible with a rapid-equilibrium random Bi Bi mechanism. The estimated constants are: for the substrates Km,acetyl-CoA 4.4 mM, Ki,acetyl-CoA 4.7 mM, Km,glutamate 8.1 mM, Ki,glutamate 8.8 mM; for the products, Ki,acetylglutamate 0.28 mM, Ki,CoA 5.6 mM. The rate constant for the forward direction is 1.24s-1. If in vivo the constants are of the same order of magnitude as in vitro, the synthesis of N-acetylglutamate, an obligate activator of the first step of urea synthesis, can be expected to occur in the mitochondrion under conditions where the amino acid acetyltransferase is not saturated by its substrates. The regulation of the first step of urea synthesis could thus depend mainly on the intramitochondrial substrate and perhaps product concentrations of amino acid acetyltransferase.     

Kinetic distinction between rapid-equilibrium random and abortive ordered enzymatic mechanisms using alternative substrates or kinetic isotope effects

Alternative substrates, such as those isotopically-labeled, which differ in their rate constants of catalysis but not in their rate constants of binding, generate identical values of V/Ka in ordered kinetic mechanisms of bireactant enzymes. This is shown to be true even for the rapid-equilibrium ordered mechanism in which an abortive complex between free enzyme and the second substrate is formed. In contrast, rapid-equilibrium random mechanisms have non-identical values for V/Ka. Consequently, the effect of alternative substrates or isotope effects on V/Ka provides a means to distinguish between these nearly identical kinetic mechanisms.     

Nonlinear steady-state kinetics of chloramphenicol acetyltransferase.

Steady-state kinetic analysis of chloramphenicol acetyltransferase showed that medium effects (higher temperatures or pH, higher ionic strengths, or lower values for dielectric constant) altered the kinetic behaviour of the enzyme with acetyl-CoA as substrate, but did not significantly affect behaviour with chloramphenicol. This was manifest as an increase in the degree of the rate equation to a 2:2 function. This is interpreted in terms of perturbations to the enzyme at or near the acetyl-CoA binding region of the enzyme.     

Evaluation of the catalytic mechanism of the p21-activated protein kinase PAK2

The p21-activated kinases (PAKs) play important roles in diverse cellular processes. In the present study, we provide an in-depth kinetic analysis of one of the PAK family members, PAK2, in phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate derived from LIM kinase, LIMKtide. Steady-state kinetic analysis of the initial reaction velocity of PAK2 phosphorylation of MBP is consistent with both randomly and compulsorily ordered mechanisms. Further kinetic studies carried out in various concentrations of sucrose revealed that solvent viscosities had no effect on k(cat)/K(m) for either ATP or MBP while k(cat) was highly sensitive to solvent viscosity, indicating that the enzymatic phosphorylation by PAK2 can be best interpreted by a rapid-equilibrium random bi-bi reaction model, and k(cat) is partially limited by both phosphoryl group transfer (31 s(-)(1)) and the product release (19 s(-)(1)). In the phosphorylation of LIMKtide, both k(cat) and k(cat)/K(m) were insensitive to solvent viscosity, and the product release (86 s(-)(1)) was much faster than the phosphoryl group transfer step (19 s(-)(1)). These studies suggest that the release of phospho-MBP product is likely partially rate determining for the PAK2-catalyzed reaction since the dissociation rate of products from the PAK2 active site for LIMKtide phosphorylation differs from that of MBP significantly. Such a mechanism is in contrast to the previously established kinetics for the phosphorylation of peptide substrates by cAMP-dependent kinase, in which this process is limited by the release of ADP but not the phospho-peptide product. These results complement previous structure-function studies of PAKs and provide important insight for mechanistic interpretation of the kinase functions.     

The kinetic mechanism of sheep liver sorbitol dehydrogenase.

The relations between the kinetic parameters for both sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase show that a Theorell-Chance compulsory order mechanism operates from pH 7.4 to 9.9. This is supported by many parallels with the kinetics of horse liver alcohol dehydrogenase, which operates by this classical mechanism. An isotope-exchange study using D-(2H8)sorbitol confirmed the existence of ternary complexes and that, under maximum velocity conditions, their interconversion is not rate-determining. Substrate inhibition at high concentrations of D-sorbitol or D-fructose confirmed rate-determining enzyme--coenzyme product dissociation, slowed by the existence of more stable abortive ternary enzyme-coenzyme product complexes with substrate. The effect of the inhibitor/activator 2,2,2-tribromoethanol showed the existence of enzyme-NAD-CBr3CH2OH complexes inhibiting the first phase of reaction and enzyme-NADH-CBr3CH2OH complexes dissociating more rapidly than the usual rate-determining enzyme-NADH coenzyme product dissociation in the final phase. Inhibition studies with dithiothreitol also confirmed an ordered binding of coenzymes and second substrates to sorbitol dehydrogenase. Neither D-sorbitol nor D-fructose had any effect on enzyme inactivation by the affinity labelling reagent DL-2-bromo-3-(5-imidazolyl)propionic acid, thus giving no evidence for their existence as binary enzyme-substrate complexes. Several alternative polyol substrates for sorbitol dehydrogenase gave the same maximum velocity as sorbitol. This indicated a common rate-limiting binary enzyme-NADH product dissociation and a similarity of mechanism. An enzyme assay for pH 7.0 and 9.9 is given which enables the concentration of sorbitol dehydrogenase to be determined from initial rate measurements of enzyme activity.     

Dependence of the substrate specificity and kinetic mechanism of horse-liver alcohol dehydrogenase on the size of the C-3 pyridinium substituent. 3-Benzoylpyridine-adenine dinucleotide

The kinetic mechanism and the substrate specificity of liver alcohol dehydrogenase are changed when 3-benzoylpyridine-adenine dinucleotide is used as coenzyme. Only primary alcohols are substrates of the enzyme and with ethanol the mechanism becomes rapid-equilibrium random bi-bi. According to model building experiments on a graphic display, the benzoyl group partially enters the substrate binding site, whereas the essential interactions between coenzyme and enzyme are preserved. This restraint on the substrate binding site provides a molecular explanation for the observed dependence between coenzyme and substrate chemical structures.