Functional dynamics in the active site of the ribonuclease binase

doi:10.1073/pnas.121069998

Functional dynamics in the active site of the ribonuclease binase

^*Biophysics Research Division, ^¶Department of Biological Chemistry, and ^‖Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109

Edited by Vincent Massey, University of Michigan Medical School, Ann Arbor, MI, and approved April 10, 2001 (received for review February 12, 2001)

Abstract

Binase, a member of a family of microbial guanyl-specific ribonucleases, catalyzes the endonucleotic cleavage of single-stranded RNA. It shares 82% amino acid identity with the well-studied protein barnase. We used NMR spectroscopy to study the millisecond dynamics of this small enzyme, using several methods including the measurement of residual dipolar couplings in solution. Our data show that the active site of binase is flanked by loops that are flexible at the 300-μs time scale. One of the catalytic residues, His-101, is located on such a flexible loop. In contrast, the other catalytic residue, Glu-72, is located on a β-sheet, and is static. The residues Phe-55, part of the guanine base recognition site, and Tyr-102, stabilizing the base, are the most dynamic. Our findings suggest that binase possesses an active site that has a well-defined bottom, but which has sides that are flexible to facilitate substrate access/egress, and to deliver one of the catalytic residues. The motion in these loops does not change on complexation with the inhibitor d(CGAG) and compares well with the maximum k _cat (1,500 s⁻¹) of these ribonucleases. This observation indicates that the NMR-measured loop motions reflect the opening necessary for product release, which is apparently rate limiting for the overall turnover.

Footnotes

↵ † Present address: Biotechnology Division, National Institute of Standards and Technology, Building 222, Mailstop 8314, Gaithersburg, MD 20899-8314.
↵ ‡ L.W. and Y.P. contributed equally to this work.
↵ § Present address: Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115.
↵ ** To whom reprint requests should be addressed. E-mail: zuiderwe{at}umich.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

CPMG,

Carr-Purcell-Meiboom-Gill;

NMA,

N-methyl acetamide;

HSQC,

heteronuclear single quantum correlation