Roles of static and dynamic domains in stability and catalysis of adenylate kinase

doi:10.1073/pnas.0507527103

Roles of static and dynamic domains in stability and catalysis of adenylate kinase

Department of Biochemistry, University of Wisconsin, Madison, WI 53706

Edited by Brian W. Matthews, University of Oregon, Eugene, OR, and approved December 22, 2005 (received for review August 30, 2005)

Abstract

Protein dynamics, including conformational switching, are recognized to be crucial for the function of many systems. These motions are more challenging to study than simple static structures. Here, we present evidence suggesting that in the enzyme adenylate kinase large “hinge bending” motions closely related to catalysis are regulated by intrinsic properties of the moving domains and not by their hinges, by anchoring domains, or by remote allosteric-like regions. From a pair of highly homologous mesophilic and thermophilic adenylate kinases, we generated a series of chimeric enzymes using a previously undescribed method with synthetic genes. Subsequent analysis of the chimeras has revealed unexpected spatial separation of stability and activity control. Our results highlight specific contributions of dynamics to catalysis in adenylate kinase. Furthermore, the overall strategy and the specific mutagenesis method used in this study can be generally applied.

Footnotes

*To whom correspondence should be addressed at:
University of Wisconsin, 433 Babcock Drive, Madison, WI 53706.
E-mail: phillips{at}biochem.wisc.edu
Author contributions: E.B. and G.N.P. designed research; E.B. performed research; E.B. analyzed data; and E.B. and G.N.P. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

AK,

adenylate kinase;

AKmeso,

AK from Bacillus subtilis;

AKthermo,

AK from Bacillus stearothermophilus;

DSC,

differential scanning calorimetry;

Tm,

thermal denaturation midpoint.