Interactions of a family 18 chitinase with the designed inhibitor HM508 and its degradation product, chitobiono- delta-lactone

Ala mutation in the +2 subsite of ChiB, led to reduced affinity for the inhibitor, showing that interactions between the phenyl group and the enzyme contribute to binding. Interestingly, a complex of enzymatically degraded HM508 with the wild type enzyme showed a chitobiono-delta- lactone bound in the -2 and -1 subsites, despite the fact that the equilibrium between the lactone and the hydroxy acid forms in solution lies far toward the latter. This shows that the active site preferentially binds the E-4 conformation of the -1 sugar, which resembles the proposed transition state of the reaction -->
Jazyk: English
Přístupová URL adresa: https://explore.openaire.eu/search/publication?articleId=od_______266::3d94d703113319b28b4abdc26eddf1b3
https://publishup.uni-potsdam.de/frontdoor/index/index/docId/15396
Rights: CLOSED
Přírůstkové číslo: edsair.od.......266..3d94d703113319b28b4abdc26eddf1b3
Autor: Vaaje-Kolstad, G., Vasella, A., Peter, Martin G., Netter, C., Houston, Douglas R., Westereng, B., Synstad, Bjoenar, Eijsink, Vincent G. H., van Aalten, Daan M. F.
Jazyk: angličtina
Rok vydání: 2004
Předmět:
Popis: We describe enzymological and structural analyses of the interaction between the family 18 chitinase ChiB from Serratia marcescens and the designed inhibitor N,N'-diacetylchitobionoxime-N-phenylcarbamate (HM508). HM508 acts as a competitive inhibitor of this enzyme with a K-i in the 50 muM range. Active site mutants of ChiB show K-i values ranging from 1 to 200 muM, providing insight into some of the interactions that determine inhibitor affinity. Interestingly, the wild type enzyme slowly degrades HM508, but the inhibitor is essentially stable in the presence of the moderately active D142N mutant of ChiB. The crystal structure of the D142N-HM508 complex revealed that the two sugar moieties bind to the -2 and -1 subsites, whereas the phenyl group interacts with aromatic side chains that line the +1 and +2 subsites. Enzymatic degradation of HM508, as well as a Trp-->Ala mutation in the +2 subsite of ChiB, led to reduced affinity for the inhibitor, showing that interactions between the phenyl group and the enzyme contribute to binding. Interestingly, a complex of enzymatically degraded HM508 with the wild type enzyme showed a chitobiono-delta- lactone bound in the -2 and -1 subsites, despite the fact that the equilibrium between the lactone and the hydroxy acid forms in solution lies far toward the latter. This shows that the active site preferentially binds the E-4 conformation of the -1 sugar, which resembles the proposed transition state of the reaction
Databáze: OpenAIRE
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