Format

Send to

Choose Destination
See comment in PubMed Commons below
Biochemistry. 2008 Dec 23;47(51):13489-96. doi: 10.1021/bi801108a.

Relative tolerance of an enzymatic molten globule and its thermostable counterpart to point mutation.

Author information

  • 1Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.

Abstract

Enzyme structures reflect the complex interplay between the free energy of unfolding (DeltaG) and catalytic efficiency. Consequently, the effects of point mutations on structure, stability, and function are difficult to predict. It has been proposed that the mutational robustness of homologous enzymes correlates with a higher initial DeltaG. To examine this issue, we compared the tolerance of a natural thermostable chorismate mutase and an engineered molten globular variant to targeted mutation. These mutases possess similar sequence, structure, and catalytic efficiency but dramatically different DeltaG values. We find that analogous point mutations can have widely divergent effects on catalytic activity in these scaffolds. In a set of five rationally designed single-amino acid changes, the thermostable scaffold suffers activity losses ranging from 50-fold smaller, for an aspartate-to-glycine substitution at the active site, to 2-fold greater, for a phenylalanine-to-tryptophan substitution in the hydrophobic core, versus that of the molten globular scaffold. However, biophysical characterization indicates that the variations in catalytic efficiency are not caused by losses of either secondary structural integrity or thermodynamic stability. Rather, the activity differences between variant pairs are very much context-dependent and likely stem from subtle changes in the fine structure of the active site. Thus, in many cases, it may be more productive to focus on changes in local conformation than on global stability when attempting to understand and predict how enzymes respond to point mutations.

PMID:
19053245
DOI:
10.1021/bi801108a
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for American Chemical Society
    Loading ...
    Support Center