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The phosphorylated intermediate in the phosphoglyceromutase reaction.

ABSTRACT: 1. High-voltage paper-electrophoresis methods have been used for the separation of (32)P-labelled phosphoesters. 2. Evidence is presented which indicates that (32)P-labelled phosphopeptides, obtained after acid hydrolysis of phosphoglyceromutase incubated with impure 2,3-di[(32)P]phosphoglycerate, are derived from phosphoglucomutase contamination. 3. The hydrolysis of 2,3-di[(32)P]phosphoglycerate by phosphoglyceromutase has been studied. After an apparent instantaneous hydrolysis of 1 mole of coenzyme/mole of enzyme the reaction proceeds at a very low rate. 4. This hydrolysis seems to be due to the destruction of an enzyme-coenzyme complex. The proportions of the decomposition products of the complex vary according to further handling (pH of ionophoresis). 5. The inorganic [(32)P]phosphate produced by the hydrolysis of the complex and the inorganic [(32)P]phosphate produced by the slow phosphatase activity can be differentiated by the ability of the former to be incorporated into non-radioactive substrate before enzyme denaturation. 6. The effect of enzyme concentration on the stoicheiometry of the slow phosphatase hydrolysis of the diphosphoglycerate is described and this suggests that it may occur via two independent reactions, one of them being the decomposition of the enzyme-coenzyme complex on standing.

SUBMITTER: Zwaig N

PROVIDER: S-EPMC1264852 | biostudies-other | 1966 Feb

REPOSITORIES: biostudies-other

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Project description:o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered bi uni uni bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first half-reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the "F" form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered bi uni uni bi iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogues with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogues tested as none were active except 4-[2-(trifluoromethyl)phenyl]-4-oxobutyric acid which exhibited a 100-fold decrease in k(cat)/K(m). On the basis of an understanding of OSB-CoA synthetase's kinetic mechanism and substrate specificity, a reaction intermediate analogue of OSB-AMP, 5'-O-{N-[2-(trifluoromethyl)phenyl]-4-oxobutyl}adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase class of enzymes.

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The phosphorylated intermediate in the phosphoglyceromutase reaction.

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