SLOW-BINDING AND FAST-BINDING INHIBITORS OF THERMOLYSIN DISPLAY DIFFERENT MODES OF BINDING - CRYSTALLOGRAPHIC ANALYSIS OF EXTENDED PHOSPHONAMIDATE TRANSITION-STATE ANALOGS
SLOW-BINDING AND FAST-BINDING INHIBITORS OF THERMOLYSIN DISPLAY DIFFERENT MODES OF BINDING - CRYSTALLOGRAPHIC ANALYSIS OF EXTENDED PHOSPHONAMIDATE TRANSITION-STATE ANALOGS
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DOI：
10.1021/bi00400a008
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发表时间：
1987-12-29
期刊：
影响因子：
2.9
通讯作者：
MATTHEWS, BW
中科院分区：
文献类型：
--
作者：
HOLDEN, HM;TRONRUD, DE;MATTHEWS, BW
两种膦酰胺肽抑制剂——苯甲氧基-GlyP-L-Leu-L-Leu (ZGPLL) 和苯甲氧基-L-PheP-L-Leu-L-Ala (ZFPLA) 与嗜热菌蛋白酶的结合模式已通过 X 射线晶体学测定，并以高分辨率精修至晶体学 R 值分别为 17.7% 和 17.0%。 （GlyP 用于指示肽键的三角碳被膦酰胺基团的四面体磷取代。）这些抑制剂被设计为假定的催化过渡态的结构类似物，并且是嗜热菌蛋白酶的有效抑制剂（ZGPLL，Ki = 9.1 nM；ZFPLA，Ki = 0.068 nM） [Bartlett, P. A. 和 Marlowe, C. K. (1987) 生物化学（本期下一篇论文）]。 ZFPLA 以过渡态预期的方式与嗜热菌蛋白酶结合，并首次为 S2 亚位点中延伸底物的假定结合模式提供直接支持。 ZFPLA 的结合模式显示了所有被认为可以稳定过渡态的相互作用，并支持假定的催化机制 [Hangauer, D. G., Monzingo, A. F., and Matthews, B. W. (1984) Biochemistry 23, 5730-5741]。膦酰胺部分的两个氧与锌配位，形成金属的整体五配位。对于第二种抑制剂，情况有所不同。尽管ZFPLA和ZGPLL在S1''和S2''亚位点上具有相似的结合模式，但苯甲氧基-Phe和苯甲氧基-Gly部分的构型不同。对于ZFPLA，苯甲氧基的羰基直接与酶形成氢键，而在ZGPLL中，羰基旋转117°，并且在抑制剂和酶之间插入水分子。对于 ZGPLL，只有一个膦酰胺氧与锌配位。与抑制剂-锌连接从ZGPLL中的单齿到ZFPLA中的双齿的变化相关，磷-氮键长增加了约0.25ANG，强烈表明ZFPLA中的磷酰胺氮是阳离子的，类似于过渡态的双质子化氮。 ZFPLA 的氮似乎向蛋白质提供两个氢键的观察结果也表明它是阳离子的。各个抑制剂采用的不同构型与其结合动力学的巨大差异相关[Bartlett, P. A., and Marlowe, C. K. (1987) Biochemistry（本期下一篇文章）]。这些动力学差异与酶的任何显着构象变化无关。相反，ZFPLA和相关α-取代抑制剂的缓慢结合行为与特定水分子从活性位点的位移相关。
The modes of binding to thermolysin of two phosphonamidate peptide inhibitors, carbobenzoxy-GlyP-L-Leu-L-Leu (ZGPLL) and carbobenzoxy-L-PheP-L-Leu-L-Ala (ZFPLA), have been determined by X-ray crystallography and refined at high resolution to crystallographic R-values of 17.7% and 17.0%, respectively. (GlyP is used to indicate that the trigonal carbon of the peptide linkage is replaced by the tetrahedral phosphorus of a phosphonamidate group.) These inhibitors were designed to be structural analogues of the presumed catalytic transition state and are potent inhibitors of thermolysin(ZGPLL, Ki = 9.1 nM; ZFPLA, Ki = 0.068 nM) [Bartlett, P. A., and Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. ZFPLA binds to thermolysin in the manner expected for the transition state and, for the first time, provides direct support for the presumed mode of binding of extended substrates in the S2 subsite. The mode of binding of ZFPLA displays all the interactions that are presumed to stabilize the transition state and supports the postulated mechanism of catalysis [Hangauer, D. G., Monzingo, A. F., and Matthews, B. W. (1984) Biochemistry 23, 5730-5741]. The two oxygens of the phosphonamidate moiety are liganded to the zinc to give overall pentacoordination of the metal. For the second inhibitor the situation is different. Although both ZFPLA and ZGPLL have similar modes of binding in the S1'' and S2'' subsites, the configurations of the carbobenzoxy-Phe and carbobenzoxy-Gly moieties are different. For ZFPLA the carbonyl group of the carbobenzoxy group is hydrogen bonded directly to the enzyme, whereas in ZGPLL the carbonyl group is rotated 117.degree., and there is a water molecule interposed between the inhibitor and the enzyme. For ZGPLL only one of the phosphonamidate oxygens is liganded to the zinc. Correlated with the change in inhibitor-zinc ligation from monodentate in ZGPLL to bidentate in ZFPLA there is an increase in the phosphorus-nitrogen bond length of about 0.25 .ANG., strongly suggesting that the phosphonamide nitrogen in ZFPLA is cationic, analogous to the doubly protonated nitrogen of the transition state. The observation that the nitrogen of ZFPLA appears to donate two hydrogen bonds to the protein also indicates that it is cationic. The different configurations adopted by the respective inhibitors are correlated with large differences in their kinetics of binding [Bartlett, P. A., and Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. These differences in kinetics are not associated with any significant conformational change on the part of the enzyme. Rather, the slow-binding behavior of ZFPLA and related .alpha.-substituted inhibitors is correlated with the displacement of a specific water molecule from the active site.