Quirk D J, Raines R T
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706 USA.
Biophys J. 1999 Mar;76(3):1571-9. doi: 10.1016/S0006-3495(99)77316-9.
Bovine pancreatic ribonuclease A (RNase A) has a conserved His ... Asp catalytic dyad in its active site. Structural analyses had indicated that Asp121 forms a hydrogen bond with His119, which serves as an acid during catalysis of RNA cleavage. The enzyme contains three other histidine residues including His12, which is also in the active site. Here, 1H-NMR spectra of wild-type RNase A and the D121N and D121A variants were analyzed thoroughly as a function of pH. The effect of replacing Asp121 on the microscopic pKa values of the histidine residues is modest: none change by more than 0.2 units. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. In the presence of the reaction product, uridine 3'-phosphate (3'-UMP), protonation of one active-site histidine residue favors protonation of the other. This finding is consistent with the phosphoryl group of 3'-UMP interacting more strongly with the two active-site histidine residues when both are protonated. Comparison of the titration curves of the unliganded enzyme with that obtained in the presence of different concentrations of 3'-UMP shows that a second molecule of 3'-UMP can bind to the enzyme. Together, the data indicate that the aspartate residue in the His ... Asp catalytic dyad of RNase A has a measurable but modest effect on the ionization of the adjacent histidine residue.
牛胰核糖核酸酶A（RNase A）在其活性位点有一个保守的His...Asp催化二元组。结构分析表明，Asp121与His119形成氢键，His119在RNA切割催化过程中作为酸起作用。该酶还含有其他三个组氨酸残基，包括也位于活性位点的His12。在此，对野生型RNase A以及D121N和D121A变体的1H-NMR光谱作为pH的函数进行了全面分析。将Asp121替换对组氨酸残基微观pKa值的影响较小：没有一个变化超过0.2个单位。没有证据表明His119与121位的天冬氨酸或天冬酰胺残基之间形成了低势垒氢键。在反应产物尿苷3'-磷酸（3'-UMP）存在的情况下，一个活性位点组氨酸残基的质子化有利于另一个的质子化。这一发现与当两个活性位点组氨酸残基都质子化时3'-UMP的磷酰基与它们的相互作用更强是一致的。将未结合配体的酶的滴定曲线与在不同浓度3'-UMP存在下获得的滴定曲线进行比较，结果表明第二个3'-UMP分子可以与该酶结合。总之，这些数据表明RNase A的His...Asp催化二元组中的天冬氨酸残基对相邻组氨酸残基的电离有可测量但较小的影响。
