Evaluation of critical structural elements of UDP-sugar substrates and certain cysteine residues of a vertebrate hyaluronan synthase.
The hyaluronan (HA) synthases catalyze the addition of two different monosaccharides from UDP-sugar substrates to the linear heteropolysaccharide chain. To accomplish this task, the HA synthases must be able to bind and to transfer from both UDP-sugar substrates. Until now, it has been impossible to distinguish between these two abilities. We have created a mutant of xlHAS1, a HA synthase from Xenopus laevis, that allows for the examination of the enzyme's ability to bind substrate only. The ability of different compounds to protect the xlHAS1(C337S) mutant enzyme from loss of activity due to treatment with N-ethylmaleimide, a cysteine-modifying reagent, yields information on the relative affinity of a variety of nucleotides and nucleotide-sugars. We have observed that the substrate binding selectivity is more relaxed than the specificity of catalytic transfer. The only attribute that appears to be absolutely required for binding is a nucleotide containing two phosphates complexed with magnesium ion. The role of certain cysteine residues in catalysis was also evaluated. Cys307 of xlHAS1 may play a role in catalysis or in maintaining structure. Mutation of Cys337 raises the UDP-GlcUA Michaelis constant (K(m)), suggesting that this residue participates in UDP-GlcUA substrate binding or in catalytic complex formation.
Pummill
Philip E
PE
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
DeAngelis
Paul L
PL
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000737:chemistry*; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D007700:Kinetics; D008565:Membrane Proteins; D009154:Mutation; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014166:Transferases; D014539:Uridine Diphosphate Sugars / Q000737:chemistry*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D014539:Uridine Diphosphate Sugars; D029867:Xenopus Proteins; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases; D003545:Cysteine; D005033:Ethylmaleimide