Stone S R, Rennex D, Wikstrom P, Shaw E, Hofsteenge J
Friedrich-Miescher-Institut, Basel, Switzerland.
Biochem J. 1992 May 1;283 ( Pt 3)(Pt 3):871-6. doi: 10.1042/bj2830871.
Peptidyldiazomethanes with proline in the P1 position were found to be competitive slow-binding inhibitors of prolyl endopeptidase. Progress-curve experiments monitoring the increase in the degree of inhibition with time indicated that the kinetic mechanism involved an initial complex that isomerized to form a tighter complex. Reversibility of the inhibited complex was demonstrated by monitoring the regain of enzyme activity after removal of free inhibitor and dilution into an assay containing competing substrate. The kinetics of the reversal of inhibition indicated a more complicated inhibitory mechanism involving more than one pathway for reversal of the tight complex. A slow-binding mechanism of inhibition has not been previously observed with peptidyldiazomethanes. Incorporation of [3H]Ac-Ala-Ala-Pro-diazomethane into prolyl endopeptidase was observed after denaturation of the inhibited complex. The peptide labelled with [3H]Ac-Ala-Ala-Pro-diazomethane was isolated and found to contain the active-site serine residue.
研究发现，P1位含有脯氨酸的肽基重氮甲烷是脯氨酰内肽酶的竞争性慢结合抑制剂。通过监测抑制程度随时间增加的进展曲线实验表明，动力学机制涉及一个初始复合物异构化为形成更紧密复合物的过程。通过监测去除游离抑制剂并稀释到含有竞争底物的测定体系后酶活性的恢复，证明了抑制复合物的可逆性。抑制作用逆转的动力学表明存在一种更复杂的抑制机制，涉及紧密复合物逆转的多条途径。此前尚未观察到肽基重氮甲烷有慢结合抑制机制。在抑制复合物变性后，观察到[3H]Ac-Ala-Ala-Pro-重氮甲烷掺入脯氨酰内肽酶。分离出用[3H]Ac-Ala-Ala-Pro-重氮甲烷标记的肽，发现其含有活性位点丝氨酸残基。
