Kravchuk A V, Zhao L, Kubiak R J, Bruzik K S, Tsai M D
Departments of Chemistry and Biochemistry, The Ohio State University, Columbus 43210, USA.
Biochemistry. 2001 May 8;40(18):5433-9. doi: 10.1021/bi002372q.
Phosphatidylinositol-specific phospholipase C (PI-PLC) has been proposed previously to employ a catalytic mechanism highly reminiscent of that of ribonuclease A (RNase A). Both catalytic sites are comprised of two histidine side chains acting as a general base-general acid pair and a phosphate-activating residue: an arginine in the case of PI-PLC and a lysine in RNase A. Despite these structural similarities, the PI-PLC reaction is slowed 10(5)-fold upon substitution of one of the phosphate nonbridging oxygen atoms with sulfur, whereas a much smaller effect is observed in the analogous RNase A reaction. Here, we report a systematic study of this property in PI-PLC, conducted by means of site-directed chemical modification of a cysteine residue replacing the arginine at position 69. The results show that mutant enzymes featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than do those with monodentate side chains. The results suggest that the bidentate nature of Arg69 is the origin of the large thio effects and stereoselectivity in PI-PLC. We propose that in addition to binding the phosphate, the function of arginine 69 is to bring the phosphate group and the 2-OH group of inositol into proximity and to induce proper alignment for nucleophilic attack, and possibly to lower the pK(a) of the 2-OH. The results presented here could be important to mechanisms of phosphoryl transfer enzymes in general, suggesting that a major part of thio effects observed in enzymatic phosphoryl transfer reactions can originate from factors other than direct interaction between a side chain and a phosphate group, and caution the use of the absolute magnitude of the thio effect as an indicator of the strength of such interactions.
磷脂酰肌醇特异性磷脂酶C（PI-PLC）先前被认为采用了一种与核糖核酸酶A（RNase A）极为相似的催化机制。两个催化位点均由作为一般碱-一般酸对的两个组氨酸侧链和一个磷酸激活残基组成：PI-PLC中的精氨酸以及RNase A中的赖氨酸。尽管存在这些结构上的相似性，但当磷酸非桥连氧原子之一被硫取代时，PI-PLC反应减慢了10^5倍，而在类似的RNase A反应中观察到的影响则小得多。在此，我们报告了对PI-PLC中这一特性的系统研究，该研究通过对取代第69位精氨酸的半胱氨酸残基进行定点化学修饰来进行。结果表明，在该位置具有双齿侧链的突变酶比具有单齿侧链的突变酶表现出显著更高的活性、更高的硫效应和更大的立体选择性。结果表明，Arg69的双齿性质是PI-PLC中硫效应大和立体选择性的根源。我们提出，除了结合磷酸外，精氨酸69的功能是使磷酸基团和肌醇的2-OH基团靠近，并诱导亲核攻击的正确排列，还可能降低2-OH的pK(a)。此处呈现的结果可能对一般磷酸转移酶的机制很重要，这表明在酶促磷酸转移反应中观察到的硫效应的主要部分可能源于侧链与磷酸基团之间直接相互作用以外的因素，并提醒人们注意将硫效应的绝对大小用作此类相互作用强度指标的做法。
