FUNCTIONAL INTERACTION AMONG CATALYTIC RESIDUES IN SUBTILISIN BPN
FUNCTIONAL INTERACTION AMONG CATALYTIC RESIDUES IN SUBTILISIN BPN
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DOI：
10.1002/prot.340070405
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发表时间：
1990-01-01
影响因子：
2.9
通讯作者：
WELLS, JA
中科院分区：
文献类型：
--
作者：
CARTER, P;WELLS, JA
丝氨酸蛋白酶，枯草杆菌蛋白酶BPN“的变体，其中催化三联体残基（Ser-221、His-64和Asp-32）被丙氨酸单独或组合取代，保留与底物N-琥珀酰基-L-Ala-L-Ala-L-Pro-L-Phe-P-硝基苯胺（sAAPF-pna）的活性，其比非酶促速率高至少103至104 [Carter，P.，威尔斯，J.A. Nature（伦敦）322：564-568，1988]。残余活性的可能来源是与Asn-155的N δ 2的氢键，其有助于稳定在野生型酶的酰胺键水解期间在四面体过渡态中产生的氧阴离子。用Gly（N155 G）代替Asn-155使sAAPF-pna的周转数（kcat）降低了150倍，而米氏常数（KM）几乎没有变化。然而，在组合N155 G和S221 A突变以得到N155 G：S221 A时，kcat实际上比S221 A酶大5倍。因此，Asn-155的催化作用取决于Ser-221的存在。N155 G：S221 A酶的残余活性（比未催化速率高104倍）不是人为产物，因为它可以被火鸡卵类粘蛋白抑制剂（OMTKY 3）的第三结构域完全抑制，其与活性位点形成强的1：1复合物。突变N155 G和S221 A分别单独地使枯草杆菌蛋白酶和OMTKY 3之间的相互作用减弱1.8和2.0 kcal/mol，并且组合地减弱2.1 kcal/mol。这与OMTKY 3抑制剂的反应性位点羰基周围的稳定相互作用的破坏一致。这些数据表明，Ser-221功能与Asp-155一起加速酰胺键水解和其他过渡态稳定的相互作用占103至104倍的残留率增强。更一般地说，这些研究说明了使用定点诱变来证明单个催化基团的能量重要性的局限性，所述催化基团的功能取决于与其它基团的相互作用。
Variants of the serine protease, subtilisin BPN'', in which the catalytic triad residues (Ser-221, His-64, and Asp-32) are replaced singly or in combination by alanine retain activities with the substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-P-nitroanilide (sAAPF-pna) that are at least 103 to 104 above the nonenzymatic rate [Carter, P., Wells, J.A. Nature (London) 322:564-568, 1988]. A possible source of the residual activity was the hydrogen bond with the N.delta.2 of Asn-155 that helps to stabilize the oxyanion generated in the tetrahedral transition state during amide bond hydrolysis by the wild-type enzyme. Replacing Asn-155 by Gly (N155G) lowers the turnover number (kcat) for sAAPF-pna by 150-fold with virtually no change in the Michaelis constant (KM). However, upon combining the N155G and S221A mutations to give N155G:S221A, kcat is actually 5-fold greater than for the S221A enzyme. Thus, the catalytic role of Asn-155 is dependent upon the presence of Ser-221. The residual activity of the N155G:S221A enzyme ($104-fold above the uncatalyzed rate) is not an artifact because it can be completely inhibited by the third domain of the turkey ovomucoid inhibitor (OMTKY3), which forms a strong 1:1 complex with the active site. The mutations N155G and S221A individually weaken the interaction between subtilisin and OMTKY3 by 1.8 and 2.0 kcal/mol, respectively, and in combination by 2.1 kcal/mol. This is consistent with disruption of stabilizing interactions around the reactive site carbonyl of the OMTKY3 inhibitor. These data suggest that Ser-221 functions together with Asp-155 to accelerate amide bond hydrolysis and that other transition state stabilizing interactions account for the residual rate enhancement of 103- to 104-fold. More generally, these studies illustrate the limitations of using site-directed mutagenesis to prove the energetic importance of a single catalytic group whose function is dependent upon the interaction with others.