J Biol Chem. Jul 18, 2003;278(29), 26952-26957.

The putative catalytic bases have, at most, an accessory role in the mechanism of arginine kinase.

Chemistry Biochemistry, Florida State University, Tallahassee, FL 32306-4380.

Arginine kinase is a member of the phosphagen kinase family that includes creatine kinase and likely shares a common reaction mechanism in catalyzing the buffering of cellular ATP energy levels. Abstraction of a proton from the substrate guanidinium by a catalytic base has long been thought to be an early mechanistic step. The structure of arginine kinase as a transition state analog complex (Zhou et al., 1998, Proc. Natl. Acad. Sci. USA 95; 8449 54) showed that Glu(sub225) and Glu(sub314) were the only potential catalytic residues contacting the phosphorylated nitrogen. In the present study, these residues were changed to Asp, Gln and Val or Ala in several single and multi-site mutant enzymes. These mutations had little impact on the substrate binding constants. The effect upon activity varied with reductions in kcat between 3000 and less than 2-fold. The retention of significant activity in some mutants contrasts with published studies of homologues and suggests that acid-base catalysis by these residues may enhance the rate, but is not absolutely essential. Crystal structures of mutant enzymes E314D at 1.9 and E225Q at 2.8 resolution showed that the precise alignment of substrates is subtly distorted. Thus, pre-ordering of substrates might be just as important as acid-base chemistry, electrostatics or other potential effects, in the modest impact of these residues upon catalysis.

PMID: 12732621 [PubMed - as supplied by publisher]

This publication is one of the several that describes a structure solved either at the Kasha Laboratory, Institute of Molecular Biophysics or in collaboration with the Institute Faculty. The data used for this structure determination came in full or part from the Macromolecular X-Ray Crystallography Facility.