Abstract
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.
Highlights
HA1 is a glycosaminoglycan composed of alternating repeats of the disaccharide [34]--D-GlcUA[33]--D-GlcNAc[13]
When xlHAS1 is treated with biotin-maleimide, the Western blot band shifts to ϳ5 kDa larger, indicating that there are ϳ10 free, readily available cysteines. xlHAS1 enzymes containing the C337S mutation were slightly more resistant than wild type enzyme to loss of HAS activity due to NEM, indicating that this residue is responsible in part for the NEM-mediated loss of activity
There are obviously other cysteine residues that are Apparent Ki values were determined for each compound by titrating each compound (0 – 4.5 mM) into the standard synthase assay; the Ki value equals the inhibitor concentration required to give 50% inhibition
Summary
This observation indicates that the substrate-binding requirements of the enzyme are more relaxed than the catalytic requirements. Protection experiments suggest that one or more cysteines might be part of or close to a putative substrate-binding site. We found that several cysteines of xlHAS1 were dispensable, but Cys307 may play a direct or a structural role
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