Effect of cysteine modifications on the activity of the 'small' Clostridium perfringens sialidase

The 'small' (43 kDa) sialidase of Clostridium perfringens is inhibited by l ow concentrations of mercury ions. For the investigation of possible functi onal roles of the enzyme's four cysteine residues at the amino acid positio ns 2, 282, 333 and 349, they were separately altered to serine by site-dire cted mutagenesis. The four mutant sialidases expressed in E. coli and purif ied by metal chelate chromatography were markedly reduced in specific activ ity when compared to the wild-type enzyme but with the exception of C282S e xhibited similar K-M-values indicating an unchanged mode of substrate bindi ng. The substrate specificity was also conserved for C2S, C282S, and C333S. Only the C349S sialidase exhibited a higher relative activity with colomin ic acid and the alpha 2,6-linked sialic acid of sialyllactose compared to t he alpha 2,3-linked isomer than the other mutants. Chemical modifications w ith the thiol-blocking reagents N-ethylmaleimide (NEM), p-chloromercuribenz oate (pCMB) and HgCl2 had little effect on the C282S sialidase, e.g., 6% in hibition by 5 mM NEM compared to reductions in activity between 65 and 90% for the wild-type and other mutant enzymes, supporting the idea that among the enzyme's cysteines, Cys-282 has the highest structural or functional si gnificance. The results also explain the higher mercury tolerance of Salmon ella typhimurium and Clostridium tertium sialidases, which have the positio ns equivalent to Cys-282 altered to Val and Thr, respectively, indicating t hat the thiol group of Cys-282, despite being situated near the active site , is not involved in catalysis.