Catalysis by Ribonuclease A Is Limited by the Rate of Substrate Association
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- 作者：Chiwook Park and Ronald T. Raines
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：April 1, 2003
- 年：2003
- 卷：42
- 期：12
- 页码：3509 - 3518
- 全文大小：118K
- 年卷期：v.42,no.12(April 1, 2003)
- ISSN：1520-4995
文摘
The value of kcat/KM for catalysis of RNA cleavage by ribonuclease (RNase) A can exceed109 M-1 s-1 in a solution of low salt concentration. This value approaches that expected for the diffusionalencounter of the enzyme and its substrate. To reveal the physicochemical constraints upon catalysis byRNase A, the effects of salt concentration, pH, solvent isotope, and solvent viscosity on catalysis weredetermined with synthetic substrates that bind to all of the enzymic subsites and thereby enable a meaningfulanalysis. The pKa values determined from pH-kcat/KM profiles at 0.010, 0.20, and 1.0 M NaCl areinconsistent with the known macroscopic pKa values of RNase A. This incongruity indicates that catalysisof RNA cleavage by RNase A is limited by the rate of substrate association, even at 1.0 M NaCl. Theeffect of solvent isotope and solvent viscosity on catalysis support this conclusion. The data are consistentwith a mechanism in which RNase A associates with RNA in an intermediate complex, which is stabilizedby Coulombic interactions, prior to the formation of a Michaelis complex. Thus, RNase A has evolvedto become an enzyme limited by physics rather than chemistry, a requisite attribute of a perfect catalyst.