Bacterial and viral sialidases: contribution of the conserved active site glutamate to catalysis.

Mutagenesis of the conserved glutamic acid of influenza type A (E277) and Micromonospora viridifaciens (E260) sialidases was performed to probe the contribution of this strictly conserved residue to catalysis. Kinetic studies of the E260D and E260C M. viridifaciens mutant enzymes reveal that the overall mechanism of action has not changed. That is, the mutants are retaining sialidases in which glycosylation and deglycosylation are rate-limiting for k(cat)/K(m) and k(cat), respectively. The solvent kinetic isotope effect and proton inventory on k(cat) for the E260C mutant sialidase provide strong evidence that the newly installed cysteine residue provides little catalytic acceleration. The results are consistent with the conserved aspartic acid residue (D92) becoming the key general acid/base residue in the catalytic cycle. In addition, the E277D mutant influenza type A sialidase is catalytically active toward 4-nitrophenyl α-D-sialoside, although no measurable hydrolysis of natural substrates was observed. Thus, mutating the glutamate residue (E277) to an aspartate increases the activation free energy of hydrolysis for natural substrates by >22 kJ/mol.

Use of conformationally restricted pyridinium alpha-D-N-acetylneuraminides to probe specificity in bacterial and viral sialidases.

Investigations into subtle changes in the catalytic activity of sialidases have been performed using enzymes from several different origins, and their results have been compared. This work highlights the potential pitfalls encountered when extending conclusions derived from mechanistic studies on a single enzyme even to those with high-sequence homology. Specifically, a panel of 5 pyridinium N-acetylneuraminides were used as substrates in a study that revealed subtle differences in the catalytic mechanisms used by 4 different sialidase enzymes. The lowest reactivity towards the artificial (pyridinium) substrates was displayed by the Newcastle disease virus hemagglutinin-neuraminidase. Moreover, in reactions involving aryl N-acetylneuraminides, the activity of the Newcastle enzyme was competitively inhibited by the 3,4-dihydro-2H-pyrano[3,2-c]pyridinium compound with a Ki = 58 micromol/L. Alternatively, the 3 bacterial enzymes tested, from Salmonella typhimurium, Clostridium perfringens, and Vibrio cholerae, were catalytically active against all members of the panel of substrates. Based on the observed effect of leaving-group ability, it is proposed that the rate-determining step for kcat (and likely for kcat/Km as well) with each bacterial enzyme is as follows: sialylation, which is concerted with conformational change for V. cholerae; and conformational change for S. typhimurium and C. perfringens.

Generation of a thermostable and denaturant-resistant peptide ligase.

The construction and characterization of a novel, thermostable, peptide ligase are described. Three amino acid substitutions were introduced into the secreted bacterial protease Streptomyces griseus protease B (SGPB). Mutations were chosen on the basis of two separate observations: (i) that a single substitution of the nucleophilic serine (S195A) created an enzyme with significant peptide-ligation activity, albeit greatly reduced stability [(2000) Chem. Biol. 7, 163], and (ii) that a pair of substitutions in the substrate-binding pocket (T213L and F228H) greatly increased the thermostability of the wild-type enzyme [(1996) J. Mol. Biol. 257, 233]. The triple mutant, named streptoligase, was found to catalyze peptide ligation (aminolysis of both a thiobenzyl ester and a p-nitroanilide-activated peptide) efficiently in nondenaturing and denaturing conditions including SDS (0.5% w/v) and guanidine hydrochloride (4.0 M). Moreover, streptoligase exhibited a half-live for unfolding of 16.3 min at 55 degrees C in the absence of stabilizing substrates. The fraction of the streptoligase-catalyzed reaction that gave coupled product with the acceptor peptide FAASR-NH(2) was greater for the p-nitroanilide donor (Sc-AAPF-pNA) than for the benzyl thioester substrate (Sc-AAPF-SBn). These observations are consistent with ligation proceeding through an acyl-enzyme intermediate involving histidine-57. In the case of the thioester donor the triple mutant promotes the direct attack of water on the thioester carbonyl carbon, in addition to hydrolysis occurring at the stage of the acyl-enzyme intermediate. The strategy of multiple point mutations outlined in this study may provide a general means of converting enzymes with chymotrypsin-like protein folds into peptide ligases.

Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase.

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile. Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide. The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose. A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised. For the wild-type enzyme, the value of the Brønsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups. Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining. The Brønsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively). These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1). Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism.