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Volume 272, Number 9, Issue of February 28, 1997 pp. 5452-5456
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Catalysis by a New Sialidase, Deaminoneuraminic Acid Residue-cleaving Enzyme (KDNase Sm), Initially Forms a Less Stable -Anomer of 3-Deoxy-D-glycero-D-galacto-nonulosonic Acid and Is Strongly Inhibited by the Transition State Analogue, 2-Deoxy-2,3-didehydro-D-glycero-D-galacto-2-nonulopyranosonic Acid, but Not by 2-Deoxy-2,3-didehydro-N-acetylneuraminic Acid

(Received for publication, August 27, 1996, and in revised form, November 3, 1996)

Takaho Terada , Ken Kitajima , Sadako Inoue ^¶ , Jennifer C. Wilson , Adele K. Norton , David C. M. Kong , Robin J. Thomson , Mark von Itzstein and Yasuo Inoue ^¶

From the  Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan, the ^¶ Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan, and the  Department of Medicinal Chemistry, Victorian College of Pharmacy, Monash University, Parkville, 3052 Victoria, Australia

Deaminoneuraminic acid residue-cleaving enzyme (KDNase Sm) is a new sialidase that has been induced and purified from Sphingobacterium multivorum. Catalysis by this new sialidase has been studied by enzyme kinetics and ¹H NMR spectroscopy. V_max/K_m values determined for synthetic and natural substrates of KDNase Sm reveal that 4-methylumbelliferyl-KDN (KDN2MeUmb, V_max/K_m = 0.033 min¹) is the best substrate for this sialidase, presumably because of its good leaving group properties. The transition state analogue, 2,3-didehydro-2,3-dideoxy-D-galacto-D-glycero-nonulosonic acid, is a strong competitive inhibitor of KDNase Sm (K_i = 7.7 µM versus K_m = 42 µM for KDN2MeUmb). 2-Deoxy-2,3-didehydro-N-acetylneuraminic acid and 2-deoxy-2,3-didehydro-N-glycolylneuraminic acid are known to be strong competitive inhibitors for bacterial sialidases such as Arthrobacter ureafaciens sialidase; however, KDNase Sm activity is not significantly inhibited by these compounds. This observation suggests that the hydroxyl group at C-5 is important for recognition of the inhibitor by the enzyme.

Reversible addition of water molecule (or hydroxide ion) to the reactive sialosyl cation, presumably formed at the catalytic site of KDNase Sm, eventually gives rise to two different adducts, the - and -anomers of free 3-deoxy-D-glycero-D-galacto-nonulosonic acid. ¹H NMR spectroscopic studies clearly demonstrate that the thermodynamically less stable -form is preferentially formed as the first product of the cleavage reaction and that isomerization rapidly follows, leading to an equilibrium mixture of the two isomers, the -isomer being the major species at equilibrium. Therefore, we propose that KDNase Sm catalysis proceeds via a mechanism common to the known exosialidases, but the recognition of the substituent at C-5 by the enzyme differs.

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