Purification, characterization, and mechanism of action of hamster HMG-CoA reductase
Abstract
A method has been developed to purify the catalytic domain of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, E.C. 1.1.1.34) to homogeneity and in high yield. Using the developed purification protocol, together with mutagenesis and in vitro and in vivo complementation experiments, the mammalian enzyme has been determined to be catalytically active as a dimer. The dimeric enzyme has two active sites located at the interface between adjacent polypeptide subunits. Each subunit contributes catalytically essential acidic amino acids to each active site, Glu$\sp{558}$ from one polypeptide and Asp$\sp{766}$ from the adjacent polypeptide. During deacylation of HMG-CoA, it is proposed that Glu$\sp{558}$ functions as a proton donor to His$\sp{865}$, which then protonates the coenzyme A anion, CoAS$\sp-$. Protonation of CoAS$\sp-$ is required for the stimulation of the reduction of mevaldehyde by coenzyme A and for enzyme turnover. Asp$\sp{766}$ is proposed to function as a general acid-base calalyst during the overall reaction.
Degree
Ph.D.
Advisors
Rodwell, Purdue University.
Subject Area
Biochemistry|Pharmacology|Molecular biology
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