Resmini M, Vigna R, Simms C, Barber N J, Hagi-Pavli E P, Watts A B, Verma C, Gallacher G, Brocklehurst K
Department of Biochemistry, Queen Mary and Westfield College, University of London, U.K.
Biochem J. 1997 Aug 15;326 ( Pt 1)(Pt 1):279-87. doi: 10.1042/bj3260279.
The hydrolyses of 4-nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate and of a new, more soluble, substrate, 4-nitrophenyl 4'-(3-aza-7-hydroxy-2-oxoheptyl)phenyl carbonate, each catalysed by a polyclonal antibody preparation elicited in a sheep by use of an analogous phosphate immunogen, were shown to adhere closely to the Michaelis-Menten equation, in accordance with the growing awareness that polyclonal catalytic antibodies may be much less heterogeneous than had been supposed. The particular value of studies on polyclonal catalytic antibodies is discussed briefly. Both the kcat and kcat/K(m) values were shown to increase with increase in pH across a pKa of approx. 9. Group-selective chemical modification studies established that the side chains of tyrosine and arginine residues are essential for catalytic activity, and provided no evidence for the involvement of side chains of lysine, histidine or cysteine residues. The combination of evidence from the kinetic and chemical modification studies and from studies on the pH-dependence of binding suggests that catalysis involves assistance to the reaction of the substrate with hydroxide ions by hydrogen-bond donation at the reaction centre by tyrosine and arginine side chains. This combination of hydrogen-bond donors appears to be a feature common to a number of other hydrolytic catalytic antibodies. High-pKa acidic side chains may be essential for the effectiveness of catalytic antibodies that utilize hydroxide ions.
用类似的磷酸盐免疫原在绵羊体内诱导产生的多克隆抗体制备物催化4-硝基苯基4'-（3-氮杂-2-氧代庚基）苯基碳酸酯和一种新的、更易溶的底物4-硝基苯基4'-（3-氮杂-7-羟基-2-氧代庚基）苯基碳酸酯的水解反应，结果表明，这些反应紧密遵循米氏方程，这与人们日益认识到多克隆催化抗体的异质性可能比原先设想的要小得多是一致的。本文简要讨论了多克隆催化抗体研究的特殊价值。结果表明，在大约9的pKa范围内，kcat和kcat/K(m)值均随pH值的升高而增加。基团选择性化学修饰研究表明，酪氨酸和精氨酸残基的侧链对催化活性至关重要，且没有证据表明赖氨酸、组氨酸或半胱氨酸残基的侧链参与其中。动力学和化学修饰研究以及结合作用pH依赖性研究的证据表明，催化作用涉及酪氨酸和精氨酸侧链在反应中心通过氢键供体作用协助底物与氢氧根离子反应。这种氢键供体的组合似乎是许多其他水解催化抗体共有的特征。高pKa酸性侧链对于利用氢氧根离子的催化抗体的有效性可能至关重要。
