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.
Watson
Jacqueline N
JN
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.
Knoll
Tara L
TL
Chen
Johnny H
JH
Chou
Doug T H
DT
Borgford
Thor J
TJ
Bennet
Andrew J
AJ
D001667:Binding, Competitive; D003016:Clostridium perfringens / Q000201:enzymology; D007700:Kinetics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009522:Newcastle disease virus / Q000201:enzymology; D011487:Protein Conformation; D011726:Pyridinium Compounds / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014734:Vibrio cholerae / Q000201:enzymology
D011726:Pyridinium Compounds; D011991:Receptors, Virus; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid