The role of Asp-295 in the catalytic mechanism of Leuconostoc mesenteroides sucrose phosphorylase probed with site-directed mutagenesis.

Abstract:

:Replacements of Asp-295 by Asn (D295N) and Glu (D295E) decreased the catalytic center activity of Leuconostoc mesenteroides sucrose phosphorylase to about 0.01% of the wild-type level (k(cat)=200s(-1)). Glucosylation and deglucosylation steps of D295N were affected uniformly, approximately 10(4.3)-fold, and independently of leaving group ability and nucleophilic reactivity of the substrate, respectively. pH dependences of the catalytic steps were similar for D295N and wild-type. The 10(5)-fold preference of the wild-type for glucosyl transfer compared with mannosyl transfer from phosphate to fructose was lost in D295N and D295E. Selective disruption of catalysis to glucosyl but not mannosyl transfer in the two mutants suggests that the side chain of Asp-295, through a strong hydrogen bond with the equatorial sugar 2-hydroxyl, stabilizes the transition states flanking the beta-glucosyl enzyme intermediate by > or = 23kJ/mol.

journal_name

FEBS Lett

journal_title

FEBS letters

authors

Mueller M,Nidetzky B

doi

10.1016/j.febslet.2007.02.060

subject

Has Abstract

pub_date

2007-04-03 00:00:00

pages

1403-8

issue

7

eissn

0014-5793

issn

1873-3468

pii

S0014-5793(07)00226-8

journal_volume

581

pub_type

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