Kinetic and mutational studies of the number of interacting divalent cations required by bacterial and human methionine aminopeptidases.

Methionine aminopeptidases (MetAP) are responsible for the proteolytic removal of the initiator methionine from nascent proteins. This processing permits multiple posttranslational modifications and protein turnover. We have cloned, expressed in Escherichia coli, and purified the recombinant human mitochondrial MetAP isoform (MetAP1D).

X-ray, NMR, and mutational studies of the catalytic cycle of the GDP-mannose mannosyl hydrolase reaction.

GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-alpha-D-hexose to GDP and beta-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (free enzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with the structure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An important loop (L6, residues 119-125) contains a ligand to the essential Mg2+ (Gln-123), the catalytic base (His-124), and three anionic residues.

Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction.

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V.

Mutational, structural, and kinetic evidence for a dissociative mechanism in the GDP-mannose mannosyl hydrolase reaction.

GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-alpha-d-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.

Half-of-the-sites binding of reactive intermediates and their analogues to 4-oxalocrotonate tautomerase and induced structural asymmetry of the enzyme.

4-Oxalocrotonate tautomerase (4-OT), a homohexameric enzyme, converts the unconjugated enone, 2-oxo-4-hexenedioate (1), to the conjugated enone, 2-oxo-3-hexenedioate (3), via a dienolic intermediate, 2-hydroxymuconate (2). Pro-1 serves as the general base, and both Arg-11 and Arg-39 function in substrate binding and catalysis in an otherwise hydrophobic active site. Although 4-OT exhibits hyperbolic kinetics and no structural asymmetry either by X-ray or by NMR, inactivation by two affinity labels showed half-site stoichiometry [Stivers, J.