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Substituted enzyme (or ping-pong) mechanisms usually involve enzymes that exist in two forms that alternate during the catalytic reaction. A method is described here for determining the position of the equilibrium of a half reaction in a ping-pong enzyme mechanism that is based on the kinetics of the burst reaction which occurs upon addition of reactants that recycle the enzyme from one form to another. The theoretical basis for the analysis is developed, and the method is applied to the half reaction of the aldimine form of aspartate transaminase with difluoro-oxaloacetate.
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The mechanism of action of the eukaryotic initiation factor eIF-2B in catalyzing the exchange of guanine nucleotides bound to eIF-2 is uncertain--evidence having been adduced for a sequential mechanism and for a substituted enzyme mechanism. Data purporting to support a substituted enzyme mechanism have been analysed and shown to be ambiguous and equally consistent with a sequential mechanism. Suitable rate constants for a sequential mechanism involving the transient formation of the quaternary complex eIF-2.eIF-2B.GDP.GTP are suggested.
The kanamycin inactivating enzyme, 3'-phosphotransferase and 6'-acetyltransferase were first found in 1967 and on the basis of the enzymic mechanism of resistance a new research approach to the development of active useful derivatives was explored. The enzymic mechanism of resistance was conclusively confirmed by the synthesis of 3'-deoxykanamycin A and 3',4'-dideoxykanamycin B which did not undergo inactivation by 3'-phosphotransferase and inhibited the growth of resistant strains. Besides APH(3') and AAC(6') described above, the following enzymes were found to be involved in the mechanism of resistance to aminoglycosides: APH(3''), APH(5''), APH(6), APH(2''), AAC(3), AAC(2'), AAD(3''), AAD(2''), AAD(4'), AAD(6). Not only the removal of the group which undergoes the enzyme reaction but also the modification of the group binding to the enzyme has also given active derivatives such as amikacin etc. The substrate specificity of the enzymes, enzymes in the immobilized state, and the application of proton and 13C nmr for structure determination of reaction products are reviewed. It was noticed that all enzymes involved in resistance contain adenosine- and aminoglycoside-binding sites. These enzymes were thus suggested to be mainly different primarily in the positional relationships between these binding sites. It suggests a close evolutionary relationships of these enzymes. The role of these enzymes in the biosynthesis of aminoglycoside antibiotics is discussed and a general mode of the biosynthesis of aminoglycosides is proposed: a gene or gene set involved in biosynthesis of 2-deoxystreptamine which has no cytotoxicity is widely distributed and the deoxystreptamine produced is transformed to the final products.
Three variants of the classical acyl-enzyme mechanism were compared theoretically with respect to the predicted transient kinetics of substrate hydrolysis by Escherichia coli alkaline phosphatase. In all three, acyl-enzyme hydrolysis was assumed to exist initially primarily as a noncovalent complex with the acid product, inorganic phosphate. In one mechanism, the pre-steady-state rate-controlling step was assumed to be the dissociation of acid product from its initial complex with enzyme. In the other two, pre-steady-state rate control was assigned to an enzyme isomerization occurring before or after substrate binding to free enzyme. Under concentration conditions of excess substrate and acid product, integrated rate laws were used to reject the possibility of pre-steady-state rate control by enzyme isomerization between phosphate dissociation and substrate binding. Whereas this mechanism predicts a pre-steady-state noncompetitive relationship between substrate and acid product, the stopped-flow kinetics of 4-methylumbelliferyl phosphate hydrolysis demonstrates a competitive relationship, consistent with either of the other two mechanisms. Under concentration conditions of stoichiometrically limiting substrate, computer simulations eliminated the possibility of rate control by enzyme isomerization after substrate binding. This mechanism predicts a substrate concentration dependence for the apparent first-order rate constant of substrate hydrolysis which disagrees with previously published data [Halford, S. E. (1971) Biochem. J. 125, 319--327]; the other two mechanisms are consistent with experiment. Comparison of transient kinetic theory and experiment under these two contrasting concentration conditions suggests strongly that the rate-controlling step in phosphate ester hydrolysis by E. coli alkaline phosphate is the dissociation of "sticky" acid product from its noncovalent complex with enzyme. This mechanism explains an anomaly in the stopped-flow kinetic trace, a substoichiometric pre-steady-state burst of alcohol product release.
Recent theoretical contributions to the elucidation of mechanisms for iron containing enzymes are reviewed. The method used in most of these studies is hybrid density functional theory with the B3LYP functional. Three classes of enzymes are considered, the mononuclear non-heme enzymes, enzymes containing iron dimers, and heme-containing enzymes. Mechanisms for both dioxygen and substrate activations are discussed. The reactions usually go through two half-cycles, where a high-valent intermediate Fe(IV)O species is created in the first half-cycle, and the substrate reactions involving this intermediate occur in the second half-cycle. Similarities between the three classes of enzymes dominate, but significant differences also exist.
The conclusion that the steady-state kinetics of isotope exchange at equilibrium do not show first-order behaviour for some one substrate-one product enzymic mechanisms in which two molecules of substrate or product can be combined with an enzyme molecule at the one time was shown to be erroneous.
The steady-state equations for "random" enzymic mechanisms (ones with alternative routes for substrate and enzyme to form enzyme-substrate complexes) are non-Michaelian and very complicated when a quasi-equilibrium approximation cannot be used. General methods for simplifying their forms and derivations are given and applied to several single-substrate mechanisms of general or topical interest. The special simplifications resulting from partial ordering of reaction mechanism, from gross inequalities of rate constants, and from special relationships between catalytic and dissociation rate constants, are considered with reference to allosteric mechanisms. Some equations mentioned, but not given here, and more detailed working out of some of those given, have been deposited as Supplementary Publication SUP 50069 (18 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 given in Biochem. J. (1976) 153, 5.
BASIC computer programs have been designed to calculate enzyme kinetic and ligand binding parameters using distribution-free methods. Statistical estimates for the parameters can be calculated, and models distinguished by the F-test. Integrated rate equations are derived for complex enzyme mechanisms.
Hydrogen transfer--an essential component of most biological reactions--is a quantum problem. A crucial question of great current interest is how enzymes modulate the quantum dynamics of hydrogen transfer to achieve their outstanding catalytic properties. That tunnelling occurs is now widely accepted, with the conceptual frameworks incorporating protein motion into the enzymic H-tunnelling process. Computational simulation can be used to help elucidate how enzymes work and facilitate H-tunnelling at the atomic level. We review the strength of a multidisciplinary approach--combining computational simulations with enzyme kinetics and structural biology--in revealing tunnelling mechanisms in enzymes. We focus on two paradigm systems--aromatic amine dehydrogenase, in which H-tunnelling is facilitated by fast (sub-picosecond) short range motions, and dihydrofolate reductase, in which a network of long-range coupled motions drives the tunnelling event.
Bacterial Delta 5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been intensively studied as a prototype for understanding an enzyme-catalyzed allylic rearrangement involving intramolecular proton transfer. Asp38 serves as a general base to abstract the proton from the steroid C4-H, which is a much stronger base than the carboxyl group of this residue. This unfavorable proton transfer requires 11 kcal/mol of energy which has to be provided by favorable interactions between catalytic residues and substrate in the course of the catalytic reaction. How this energy is provided at the active site of KSI has been a controversial issue, and inevitably the enzyme mechanism is not settled. To resolve these issues, we have determined the crystal structure of this enzyme at 2.3 A resolution. The crystal structure revealed that the active site environment of P. testosteroni KSI is nearly identical to that of Pseudomonas putida KSI, whose structure in complex with a reaction intermediate analogue we have determined recently. Comparison of the two structures clearly indicates that the two KSIs should share the same enzyme mechanism involving the stabilization of the dienolate intermediate by the two direct hydrogen bonds to the dienolate oxyanion, one from Tyr14 OH and the other from Asp99 COOH. Mutational analysis of the two residues and other biochemical data strongly suggest that the hydrogen bond of Tyr14 provides the more significant contribution than that of Asp99 to the requisite 11 kcal/mol of energy for the catalytic power of KSI.
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A novel electrostatic approach to the manner in which enzymes catalyze reactions is developed. In this development, the author's interpretations of, and additions to, the late K. Fajans' theory of electronic structure (referred to as the Quanticule theory of chemical binding) are presented. This theory is based upon electron densities, in analogy to density functional theory. It is used to derive formulations of molecular structure, (Fajans' formulations) which show the charges of the atomic components. These charges are shown to be reduced by polarization to what are commonly referred to as partial charges. These formulations relate to the exchange of charged atomic components during reactions. The relevance of the formulations to the catalytic activity of enzymes is illustrated with carbonic anhydrase. When viewed in terms of Fajans' formulations, the active site is seen to consist of an array of charged atoms. The positive and negative charges tend to alternate. When closely positioned, these charges provide a basis for drawing atomic components of the molecules, which are exchanged in a given reaction, into the new positions. The charges are shown to be related to the binding of substrates to the enzyme and the positioning of them so that they can interact, to the reaction itself, to an essential proton transfer, and to the dissociation of the product from the enzyme. This more extensive scope of electrostatic interactions provides a more simple view of the manner in which the catalysis is accomplished. Such catalysis is consistent with a number of other proposals about enzyme mechanisms, and appears to be applicable to a large majority of enzyme-catalyzed reactions.
The kinetic parameters of ten different enzymic mechanisms in which bimolecular transfer reactions occur concomitantly with the hydrolysis of the donor molecule have been studied. The usefulness of these parameters for making a choice of mechanism is discussed. The analysis has been extended to the use of alternative substrates in bimolecular transfer reactions that proceed without the hydrolysis of the donor molecule.