Park Chiwook, Raines Ronald T
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Biochemistry. 2003 Apr 1;42(12):3509-18. doi: 10.1021/bi026076k.
The value of k(cat)/K(M) for catalysis of RNA cleavage by ribonuclease (RNase) A can exceed 10(9) M(-1) s(-1) in a solution of low salt concentration. This value approaches that expected for the diffusional encounter of the enzyme and its substrate. To reveal the physicochemical constraints upon catalysis by RNase A, the effects of salt concentration, pH, solvent isotope, and solvent viscosity on catalysis were determined with synthetic substrates that bind to all of the enzymic subsites and thereby enable a meaningful analysis. The pK(a) values determined from pH-k(cat)/K(M) profiles at 0.010, 0.20, and 1.0 M NaCl are inconsistent with the known macroscopic pK(a) values of RNase A. This incongruity indicates that catalysis of RNA cleavage by RNase A is limited by the rate of substrate association, even at 1.0 M NaCl. The effect of solvent isotope and solvent viscosity on catalysis support this conclusion. The data are consistent with a mechanism in which RNase A associates with RNA in an intermediate complex, which is stabilized by Coulombic interactions, prior to the formation of a Michaelis complex. Thus, RNase A has evolved to become an enzyme limited by physics rather than chemistry, a requisite attribute of a perfect catalyst.
在低盐浓度溶液中，核糖核酸酶（RNase）A催化RNA裂解的k(cat)/K(M)值可超过10(9) M(-1) s(-1)。该值接近酶与其底物扩散相遇所预期的值。为揭示RNase A催化过程中的物理化学限制，使用能与所有酶亚位点结合的合成底物测定了盐浓度、pH、溶剂同位素和溶剂粘度对催化作用的影响，从而能够进行有意义的分析。在0.010、0.20和1.0 M NaCl条件下，从pH-k(cat)/K(M)曲线确定的pK(a)值与RNase A已知的宏观pK(a)值不一致。这种不一致表明，即使在1.0 M NaCl条件下，RNase A催化RNA裂解也受底物结合速率的限制。溶剂同位素和溶剂粘度对催化作用的影响支持了这一结论。数据与一种机制一致，即RNase A在形成米氏复合物之前，先与RNA形成一个由库仑相互作用稳定的中间复合物。因此，RNase A已进化成为一种受物理而非化学限制的酶，这是完美催化剂的必要属性。
