Pig liver phosphomevalonate kinase: Kinetic mechanism
Section snippets
Chemicals
ATP, PEP, NADH, ADP, EDTA, rabbit muscle pyruvate kinase, dl-mevalonic acid lactone, 2-mercaptoethanol, PPO and Tris were obtained from Sigma. DTT was supplied by Calbiochem and lactate dehydrogenase by Boehringer-Mannheim. Glycerol, formic acid, ammonium sulfate, and POPOP were obtained from Merck. [2-14C]–dlmevalonate and [8-14C]ADP were provided by New England Nuclear. All other reagents used were of analytical grade.
Chromatographic materials were obtained as follows: DEAE-cellulose DE-52
Determination of kinetic parameters
The enzyme shows hyperbolic kinetics towards both substrates. Spectrophotometric assays were performed varying the [ATP] in the range of 0.05–1.03 mM and that of MVAP between 0.026 and 0.403 mM.
Fig. 2 shows a Lineweaver–Burk plot of the results obtained using MVAP as the fixed substrate and ATP as the variable substrate. These results suggest that the enzyme follows a sequential mechanism (according to the nomenclature of Cleland [15]), in agreement with previous data [2]. The values of Km for
Discussion
The main purpose of this work has been to study the kinetic mechanism of PMK and propose a model compatible with the information available. In order to achieve this, several aspects were considered: (1) optimization of the spectrophotometric assay used for measuring PMK activity; (2) determination of the true kinetic constants of the enzyme under the optimized conditions; (3) use of product inhibition to establish the type of mechanism involved; and (4) a confirmation of the proposed mechanism
Acknowledgments
The authors thank Drs. Sergio Bazaes and Ana Maria Jabalquinto for valuable suggestions during the course of this work.
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