Functional dynamics in the active site of the ribonuclease binase

Binase, a member of a family of microbial guanyl-specific ribonucleases, ca talyzes the endonucleotic cleavage of single-stranded RNA. It shares 82% am ino acid identity with the well-studied protein barnase. We used NMR spectr oscopy to study the millisecond dynamics of this small enzyme, using severa l methods including the measurement of residual dipolar couplings in soluti on. Our data show that the active site of binase is flanked by loops that a re flexible at the 300-mus time scale. One of the catalytic residues, His-1 01, is located on such a flexible loop. In contrast, the other catalytic re sidue, Glu-72, is located on a beta -sheet, and is static. The residues Phe -55, part of the guanine base recognition site, and Tyr-102, stabilizing th e 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 f lexible to facilitate substrate access/egress, and to deliver one of the ca talytic residues. The motion in these loops does not change on complexation with the inhibitor d(CGAC) and compares well with the maximum k(cat) (1,50 0 s(-1)) Of these ribonucleases. This observation indicates that the NMR-me asured loop motions reflect the opening necessary for product release, whic h is apparently rate limiting for the overall turnover.