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Most general biochemistry textbooks present enzyme inhibition by showing how the basic Michaelis-Menten parameters K[subscript m] and V[subscript max] are affected mathematically by a particular type of inhibitor. This approach, while mathematically rigorous, does not lend itself to understanding how inhibition patterns are used to determine the kinetic aspects of an enzyme. The discussion here describes a qualitative approach to teaching enzyme inhibition that allows for a physical or mechanistic understanding. This qualitative approach to enzyme inhibition starts by recognizing that the two fundamental kinetic parameters of an enzyme catalyzed reaction are V[subscript max] and V[subscript max]/K[subscript m], which correspond to the apparent rates of reaction at very high and very low concentrations of substrate, respectively. It just so happens that the reciprocals of V[subscript max] and V[subscript max]/K[subscript m] correspond to the y-intercept and slope of the Lineweaver-Burk plot, respectively. Thus, an inhibitor that affects the y-intercept binds to the enzyme at very high substrate concentrations, and thus binds to the enzyme-substrate complex, while an inhibitor that affects the slope binds to the enzyme at very low substrate concentrations, and thus binds only to free enzyme. These simple precepts can be used to interpret the basic inhibition patterns, competitive, uncompetitive and noncompetitive, and more importantly, derive mechanistic information, especially in multisubstrate reactions. The application of these principles is illustrated by using an example from cancer chemotherapy, the inhibition of thymidylate synthase by 5-fluorouracil and leucovorin. (Contains 5 figures and 4 footnotes.)

Carboxyltransferase is one component of the multifunctional enzyme acetyl-CoA carboxylase which catalyzes the first committed step in fatty acid biosynthesis. Carboxyltransferase is an α2β2 heterotetramer and possesses two distinct but integrated functions. One function catalyzes the transfer of carbon dioxide from biotin to acetyl-CoA, whereas the other involves binding to the mRNA encoding both subunits. When carboxyltransferase binds to the mRNA both enzymatic activity and translation of the mRNA are inhibited. However, the substrate acetyl-CoA competes with mRNA for binding. Thus, mRNA binding by carboxyltransferase provides an effective mechanism for regulating enzymatic activity and gene expression. This conceptual review takes the position that regulation of enzymatic activity and gene expression of carboxyltransferase by binding to its own mRNA is at its most fundamental level the result of the symmetry in the chemical reaction catalyzed by the enzyme. The chemical reaction is symmetrical in that both substrates generate enolate anions during the course of catalysis. The chemical symmetry led to a structural symmetry in the enzyme where both the α and β subunits contain oxyanion holes that stabilize the enolate anions. Then the region of the mRNA that codes for the oxyanion holes provided the binding sites for carboxyltransferase. Thus, the symmetry of the chemical reaction formed the foundation for the evolution of the mechanism for regulation of carboxyltransferase.

The rise of antibacterial-resistant bacteria is a major problem in the United States of America and around the world. Millions of patients are infected with antimicrobial resistant bacteria each year. Novel antibacterial agents are needed to combat the growing and present crisis. Acetyl-CoA carboxylase (ACC), the multi-subunit complex which catalyses the first committed step in fatty acid synthesis, is a validated target for antibacterial agents. However, there are at present, no commercially available antibiotics that target ACC. Ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulfonylamino]benzoate (SABA1) is a compound that has been shown to have antibacterial properties against Pseudomonas aeruginosa and Escherichia coli. SABA1 inhibits biotin carboxylase (BC), the enzyme that catalyses the first half reaction of ACC. SABA1 inhibits BC via an atypical mechanism. It binds in the biotin binding site in the presence of ADP. SABA1 represents a potentially new class of antibiotics that can be used to combat the antibacterial resistance crisis. [ABSTRACT FROM AUTHOR]

Research has been conducted on the biotin carboxylase from Escherichia coli. The mechanism for the biotin deprotonation has been investigated.

Research was conducted to examine a mechanism for the study of mutants of biotin carboxylase that are free of contamination from wild-type enzyme obtained from the chromosomal copy of the gene. The role of alanine-scanning mutagenesis of residues E211, E288, N290 and R292 on reactions catalyzed by biotin carboxylase was also investigated. Results demonstrate that the ability of biotin to increase the activity of the enzyme was abolished by the mutations.

The x-ray structure of the biotin carboxylase component Acetyl-CoA carboxylase, obtained from Escherichia coli by a combination of electron density and multiple isomorphous replacement techniques, consists of three structural domains. The Acetyl-CoA carboxylase is a multicomponent enzyme having biotin carboxylase activity, carboxyltransferase functionality and a carrier protein. The N-terminal, B-domain and C-terminal constitute the structural domains.

A method for determining the O-18 isotope effects on decarboxylation reactions are developed by the application high vacuum technique. The method does not require the process of remote labelling, but an attendant synthesis is essential. The influence of O-18 on decarboxylation reactions are determined, with an accuracy equal to that provided by a mass spectrometer.

The roles of acidic and basic amino acid side chains in the reactions catalyzed by aspartate transcarbamoylase (ATCase) were examined. Four ionizing amino acid residues were determined to be involved in substrate binding and catalysis. A protonated residue with a pK value of 9 was found to be important in carbamoyl phosphate-binding activities. A residue with a pK value of 7.3 must be protonated for binding of aspartate and its analogues. Analysis of the profile of log V against pH suggested the involvement of a deprotonated group with a pK value of 7.2 and a protonated residue with a pK value of 9.5 in ATCase catalysis.

The role of Thr 55 in the catalytic mechanism of aspartate transcarbamoylase (ATCase) was examined by site-directed mutagenesis. Heavy-atom isotope effects and steady-state kinetic parameters were measured to determine the mutant catalytic trimer of ATCase containing alanine instead of Thr 55. The mutation resulted in a 2-fold decrease in binding of carbamoyl phosphate (CbmP). However, substrate affinities of the T55 mutant to aspartate and succinate remained unaffected. Results also suggested that a rate-determining step in the reaction involved a conformational change in ATCase following the formation of a Michaelis constant complex.

The role of His 134 in the catalytic mechanism of aspartate transcarbamoylase (ATCase) was investigated. His 134 in the catalytic trimer of ATCase was replaced by alanine which resulted in reduced affinities for carbamoyl phosphate, aspartate and the competitive inhibitor, succinate. However, similarities in the pK values for both mutant and wild-type enzymes suggested that protonated His 134 does not participate in the binding and catalytic activities of the enzyme between pH 6.2 and pH 9.2. Results also indicated that His 134 participated in both chemical reaction and substrate binding of the enzyme.

Carbamyl phosphate was used to investigate whether the catalysis by aspartate transcarbamylase proceed through a cyanic acid or tetrahedral intermediate. 15N kinetic isotope effects of the decomposition of the mono- and dianion of carbamyl phosphate revealed that the catalysis went by a tetrahedral intermediate. The 31P NMR spectra of the decomposition catalyzed by wild-type and H134A catalytic trimers were also presented.

The molecular dynamics (MD) simulations were used to study residues in and surrounding the active site and domain movements to gain insight into the role of catalytic mechanism of wild type Escherichia coli biotin carboxylase. The results obtained for the active-site interactions and dynamics of WT biotin carboxylase provide the insight into the design and interpretation of further experiments.

A study presenting the structure of the bacterial carboxyltransferase (CT), subunits of acetyl-coA carboxylase, from two prevalent nosocomial pathogens, Staphylococcus aureus and Escherichia coli, at a resolution of 2.0 and 3.0 Armstrong respectively was conducted. The results of both structures revealed a small, independent zinc-binding domain that lacks a complement in the primary sequence or structure of the eukaryotic homologue.