title: The kinetic mechanism of phosphomevalonate kinase.
abstract: Phosphomevalonate kinase catalyzes an essential step in the so-called mevalonate pathway, which appears to be the sole pathway for the biosynthesis of sterols and other isoprenoids in mammals and archea. Despite the well documented importance of this pathway in the cause and prevention of human disease and that it is the biosynthetic root of an enormous diverse class of metabolites, the mechanism of phosphomevalonate kinase from any organism is not yet well characterized. The first structure of a phosphomevalonate kinase from Streptococcus pneumoniae was solved recently. The enzyme exhibits an atypical P-loop that is a conserved defining feature of the GHMP kinase superfamily. In this study, the kinetic mechanism of the S. pneumoniae enzyme is characterized in the forward and reverse directions using a combination of classical initial-rate methods including alternate substrate inhibition using ADPbetaS. The inhibition patterns strongly support that in either direction the substrates bind randomly to the enzyme prior to chemistry, a random sequential bi-bi mechanism. The kinetic constants are as follows: k(cat(forward)) = 3.4 s(-1), K(i(ATP)) = 137 microm, K(m(ATP)) = 74 microm, K(i(pmev)) = 7.7 microm, K(m(pmev)) = 4.2 microm; k(cat(reverse)) = 3.9 s(-1), K(i(ADP)) = 410 microm, K(m(ADP)) = 350 microm, K(i(ppmev)) = 14 microm, K(m(ppmev)) = 12 microm, where pmev and ppmev represent phosphomevalonate and diphosphomevalonate, respectively.
authors.affiliation: Department of Biochemistry, The Albert Einstein College of Medicine, Bronx, New York 10461-1926, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Pilloff
authors.forename: Kristina
authors.initials: K
authors.lastname: Dabovic
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Romanowski
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Bonanno
authors.forename: Mary
authors.initials: M
authors.lastname: Doherty
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Burley
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Leyh
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase