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Methionine‐141 directly influences the binding of 4‐methylpyrazole in human σσ alcohol dehydrogenase
Author(s) -
Xie Peiguang T.,
Hurley Thomas D.
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.12.2639
Subject(s) - alcohol dehydrogenase , isozyme , stereochemistry , enzyme , chemistry , mutant , binding site , methionine , dehydrogenase , mutagenesis , residue (chemistry) , biochemistry , amino acid , gene
Pyrazole and its 4‐alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human σσ isoenzyme exhibits a 580‐fold lower affinity for 4‐methylpyrazole than does the human β 1 β 1 isoenzyme, with which it shares 69% sequence identity. In this study, structural and kinetic studies were utilized in an effort to identify key structural features that affect the binding of 4‐methylpyrazole in human alcohol dehydrogenase isoenzymes. We have extended the resolution of the human σσ alcohol dehydrogenase (ADH) isoenzyme to 2.5 Å resolution. Comparison of this structure to the human β 1 β 1 isoenzyme structure indicated that the side‐chain position for Met141 in σσ ADH might interfere with 4‐methylpyrazole binding. Mutation of Met141 in σσ ADH to Leu (σσ141L) lowers the K i for 4‐methylpyrazole from 350 to 10 μM, while having a much smaller effect on the K i for pyrazole. Thus, the mutagenesis results show that the residue at position 141, which lines the substrate‐binding pocket at a position close to the methyl group of 4‐methylpyrazole, directly affects the binding of the inhibitor. To rule out nonspecific structural changes due to the mutation, the X‐ray structure of the σ141L mutant enzyme was determined to 2.4 Å resolution. The three‐dimensional structure of the mutant enzyme is identical to the wild‐type enzyme, with the exception of the residue at position 141. Thus, the differences in 4‐methylpyrazole binding between the mutant and wild‐type σσ ADH isoenzymes can be completely ascribed to the local changes in the topology of the substrate binding site, and provides an explanation for the class‐specific differences in 4‐methylpyrazole binding to the human ADH isoenzymes.