Mark B L, Vocadlo D J, Zhao D, Knapp S, Withers S G, James M N
Department of Biochemistry, Canadian Institutes of Heath Research Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 2001 Nov 9;276(45):42131-7. doi: 10.1074/jbc.M107154200. Epub 2001 Aug 24.
Azasugar inhibitors of the isofagomine class are potent competitive inhibitors of configuration-retaining beta-glycosidases. This potency results from the formation of a strong electrostatic interaction between a protonated endocyclic nitrogen at the "anomeric" center of the inhibitor and the catalytic nucleophile of the enzyme. Although the majority of retaining beta-glycosidases use a mechanism involving a carboxylate residue as a nucleophile, Streptomyces plicatus beta-N-acetylhexos-aminidase (SpHEX) and related family 20 glycosidases lack such a catalytic residue and use instead the carbonyl oxygen of the 2-acetamido group of the substrate as a nucleophile to "attack" the anomeric center. Thus, a strong electrostatic interaction between the inhibitor and enzyme is not expected to occur; nonetheless, the 1-N-azasugar (2R,3R,4S,5R)-2-acetamido-3,4-dihydroxy-5-hydroxymethyl-piperidinium hydrochloride (GalNAc-isofagomine.HCl), which was synthesized and assayed for its ability to inhibit SpHEX, was found to be a potent competitive inhibitor of the enzyme (K(i) = 2.7 microm). A crystallographic complex of GalNAc-isofagomine bound to SpHEX was solved and refined to 1.75 A and revealed that the lack of a strong electrostatic interaction between the "anomeric" center of GalNAc-isofagomine and SpHEX is compensated for by a novel 2.8-A hydrogen bond formed between the equatorial proton of the endocyclic nitrogen of the azasugar ring and the carboxylate of the general acid-base residue Glu-314 of SpHEX. This interaction appears to contribute to the unexpected potency of GalNAc-isofagomine toward SpHEX.
异夫糖胺类氮杂糖抑制剂是构型保留型β-糖苷酶的强效竞争性抑制剂。这种强效性源于抑制剂“异头”中心的质子化内环氮与酶的催化亲核试剂之间形成的强静电相互作用。尽管大多数构型保留型β-糖苷酶采用涉及羧酸盐残基作为亲核试剂的机制，但褶皱链霉菌β-N-乙酰己糖胺酶（SpHEX）及相关的20家族糖苷酶缺乏这样的催化残基，而是使用底物2-乙酰氨基基团的羰基氧作为亲核试剂来“攻击”异头中心。因此，预计抑制剂与酶之间不会发生强静电相互作用；尽管如此，合成并检测了其抑制SpHEX能力的1-N-氮杂糖（2R,3R,4S,5R）-2-乙酰氨基-3,4-二羟基-5-羟甲基-哌啶盐酸盐（GalNAc-异夫糖胺·HCl）被发现是该酶的强效竞争性抑制剂（Ki = 2.7 μM）。解析并精修了GalNAc-异夫糖胺与SpHEX结合的晶体复合物至1.75 Å，结果显示GalNAc-异夫糖胺的“异头”中心与SpHEX之间缺乏强静电相互作用，这通过氮杂糖环内环氮的赤道质子与SpHEX的一般酸碱残基Glu-314的羧酸盐之间形成的新型2.8 Å氢键得到补偿。这种相互作用似乎有助于GalNAc-异夫糖胺对SpHEX产生意外的强效性。
