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Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2‐dehydrogenase
Author(s) -
Lucas James E.,
Siegel Justin B.
Publication year - 2015
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 - 10.1002/pro.2669
Subject(s) - active site , conserved sequence , mutagenesis , chemistry , biochemistry , pseudomonas fluorescens , site directed mutagenesis , enzyme , biology , peptide sequence , genetics , bacteria , mutation , gene , mutant
Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2‐dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses.