Elizabeth Heyde

Glutamate dehydrogenase of lupin nodules: kinetics of the amination reaction.

Abstract Steady state velocity studies and inhibition studies with products and substrate analogs have been conducted on the amination reaction of glutamate dehydrogenase ( l -glutamate: NAD + oxidoreductase (deaminating) EC 1.4.1.2) from lupin nodules. The results are qualitatively and quantitatively consistent with a fully ordered mechanism in which NADH binds to the enzyme first followed by 2-oxoglutarate and then NH 4 + , and in which the product l -glutamate is released before NAD + . This mechanism is the only plausible mechanism consistent with the results of studies on the reaction in both directions.

A stochastic approach to a one substrate, one product enzyme reaction in the initial velocity phase

A stochastic analysis is made of the initial phase of a one substrate, one product enzyme system. A time-dependent solution is derived for the joint probability distribution of free enzyme and product, and from this, and the conservation equations, the distributions of the other components of the reaction can be deduced. The results are valid while the amount of product released is sufficiently small for the reverse reaction between product and enzyme to be neglected. The time-dependent joint probability distribution of free enzyme and the number of interactions of free enzyme with substrate is also obtained. This can be used to indicate what proportion of the total number of substrate molecules which react with enzyme are finally converted to product molecules.

Studies on inosine monophosphate dehydrogenase. Steady state kinetics

The reaction catalyzed by IMP dehydrogenase (IMP: NAD+ oxidoreductase EC 1.2.1.14) from Aerobacter aerogenes has been investigated kinetically at pH 8.1 as a three reactant system by means of steady-state velocity studies in the absence of products, as well as by inhibition studies using products and substrate analogues. The mechanism appears to be a partially random one in which IMP and K+ can bind randomly to the free enzyme while NAD does not react unless K+ or both K+ and IMP are present on the enzyme. While the steady-state velocity data can be analysed adequately on the basis that rapid equilibrium conditions apply, this is only an approximate description of the mechanism since product inhibition studies indicate that there is a significant concentration of an enzyme-XMP (enzyme-K-XMP) complex in the steady-state.

Difficulties encountered with an ultrafiltration method for measuring the binding of ligands to protein

Abstract The ultrafiltration method of Paulus has been used to study the binding of aspartate and ITP to the catalytic subunit of aspartate transcarbamylase. Markedly different estimates of dissociation constants and of the number of moles of ligand bound per mole of enzyme have been obtained using different batches of UM 10 Diaflo membranes. Estimates derived using Visking membranes are presented for comparison. For both an amino acid and a nucleotide invalid results have been obtained using UM 10 Diaflo membranes in conjunction with this enzyme. The importance of excluding artifacts in any system studied by this method is emphasized.

Stochastic fluctuations in a one substrate one product enzyme system: Are they ever relevant?

Abstract The stochastic fluctuations in the initial phase of a one substrate one product enzyme system have been calculated for an actual enzyme reaction, using the model of Heyde & Heyde (1969). The experimental data are for the reaction catalysed by triose phosphate isomerase. It is shown that for this system the stochastic fluctuations in free enzyme, enzyme-substrate or enzyme-product complex, substrate and product are negligible. Using a more general stochastic model it is shown that stochastic fluctuations will in general be negligible for such systems except at the beginning of the transient phase.

Studies on inosine monophosphate dehydrogenase. An associating-dissociating system.

The techniques of polyacrylamide gel electrophoresis, sedimentation velocity and frontal analysis on Sephadex have been used to demonstrate that preparations of IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14) from Aerobacter aerogenes consist of a mixture of molecular weight isomers. Further, it has been shown that dissociation of the higher molecular weight forms is promoted by urea, sodium dodecyl sulphate and dithiothreitol. Under conditions comparable to those used for kinetic analyses, the enzyme has a molecular weight of about 86000 and this is the smallest active species that has been observed. In the absence of a reducing agent, the enzyme undergoes polymerization and is devoid of catalytic activity. From the amino acid composition and peptide map, it appears that the molecule with a molecular weight of 86000 is made up of two identical polypeptide chains.

Studies on the inhibition of ATP: Creatine phosphotransferase by NaCl

Abstract Kinetic experiments have been undertaken to determine the apparent inhibition constants for the reaction of Cl − with various forms of creatine kinase. The conditions wer similar to those previously used for initial-velocity and isotope-exchange studies of the reaction. It has been shown that NaCl is a non-competitive inhibitor with respect to both substrates of the forward and reverse reactions. With the lower concentrations of the inhibitor used in studying the forward reaction, both the slopes and vertical intercepts of double-reciprocal plots as appeared to be linear functions of the concentration of NaCl. On the other hand, with the higher concentrations of NaCl used to inhibit the reverse reaction, the non-competitive inhibition was of the sloep-parabolic, intercept-linear type. From the values obtained for the apparent inhibition constants, it appears that Cl − may well react at sites on the enzyme at which substrate normally combines, and that the presence of a substrate on the enzyme affects the combination of Cl − . Whereas the interaction of Cl − with the ternary enzyme complex is very weak, two Cl − can react with free enzyme. The inhibition of the reaciton by sodium acetate is considerably less than that by NaCl.

A common active site model for catalysis by chorismate mutase--prephenate dehydrogenase.

Abstract Comparison of the calculated structures for the transition states of the two reactions catalysed by chorismate mutase prephenate dehydrogenase suggests that both reactions could be catalysed at a common active site. Kinetic data for the enzyme from Aerobacter aerogenes are consistent with this possibility. On the basis of these theoretical and experimental data a model for a common active site is developed. In the model, the transition state for each reaction is bound to the enzyme via both of the two substrate carboxyl groups, and can also interact with the coenzyme nicotinamide adenine dinucleotide through a hydrogen bond between the amide moiety of the nicotinamide ring and the hydroxyl group of the substrate. Chorismate, prephenate and 4-hydroxyphenylpyruvate in their ground states form the same hydrogen bond to the coenzyme, but are bound to the enzyme via a single carboxyl group only. The additional bond formed between the enzyme and the transition state structures thus provides the transition state stabilization required for catalysis of both reactions.

A unifying concept for the active site region in aspartate transcarbamylase.

Recent investigations on the aspartate transcarbamylases (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) of Escherichia coli and Streptococcus faecalis indicate that there is a site on each enzyme, apart from the active site, at which anions can bind. It is suggested in this paper that the location of such an anion binding site on the E. coli enzyme may be directly adjacent to the part of the active site at which carbamyl phosphate binds. This hypothesis is based on data demonstrating a lack of correlation between spectral changes and kinetic effects, and on a new interpretation of results obtained with N-(phosphonacetyl)-L-aspartate, which has previously been considered to act as a transition state analogue. Such a hypothesis could explain other puzzling observations made on the catalytic subunit of this enzyme, including the dependence of substrate inhibition by aspartate on the nature of the second substrate, and the ease of formation of a dead-end enzyme-aspartate-carbamyl-aspartate complex.

Interaction between polypeptide chains within the catalytic subunit of aspartate transcarbamylase

Abstract The catalytic subunit of aspartate transcarbamylase, which normally exhibits Michaelis-Menten kinetics, gives rise to nonlinear double reciprocal plots of initial velocity data in the presence of the inhibitor ITP. The results of kinetic and binding studies on the enzyme in the presence of ITP are considered in conjunction with the fact that the catalytic subunit is composed of three polypeptide chains. The data are consistent wih the proposition that the combination of carbamyl phosphate on one polypeptide chain can hinder the combination of ITP on other polypeptide chains, and vice versa . Thus the isolated catalytic subunit is capable of exhibiting a type of cooperative effect, although this effect is only observed in the presence of the inhibitor.