Escherichia coli Methionine Aminopeptidase: Implications of Crystallographic Analyses of the Native, Mutant, and Inhibited Enzymes for the Mechanism of Catalysis
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- 作者：W. Todd Lowther ; Allen M. Orville ; David T. Madden ; Sejin Lim ; Daniel H. Rich ; and Brian W. Matthews
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：June 15, 1999
- 年：1999
- 卷：38
- 期：24
- 页码：7678 - 7688
- 全文大小：331K
- 年卷期：v.38,no.24(June 15, 1999)
- ISSN：1520-4995
文摘
By improving the expression and purification of Escherichia coli methionine aminopeptidase(eMetAP) and using slightly different crystallization conditions, the resolution of the parent structure wasextended from 2.4 to 1.9 Å resolution. This has permitted visualization of the coordination geometry andsolvent structure of the active-site dinuclear metal center. One solvent molecule (likely a 
-hydroxide)bridges the trigonal bipyramidal (Co1) and octahedral (Co2) cobalt ions. A second solvent (possibly ahydroxide ion) is bound terminally to Co2. A monovalent cation binding site was also identified about 13Å away from the metal center at an interface between the two subdomains of the protein. The first structureof a substrate-like inhibitor, (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L-Leu-L-Val-L-Phe-OMe, bound toa methionine aminopeptidase, has also been determined. This inhibitor coordinates the metal center throughfour interactions as follows: (i) ligation of the N-terminal (3R)-nitrogen to Co2, (ii, iii) bridging coordinationof the (2S)-hydroxyl group, and (iv) terminal ligation to Co1 by the keto oxygen of the pseudo-peptidelinkage. Inhibitor binding occurs with the displacement of two solvent ligands and the expansion of thecoordination sphere of Co1. In addition to the tetradentate, bis-chelate metal coordination, the substrateanalogue forms hydrogen bonds with His79 and His178, two conserved residues within the active site ofall MetAPs. To evaluate their importance in catalysis His79 and His178 were replaced with alanine. Bothsubstitutions, but especially that of His79, reduce activity. The structure of the His79Ala apoenzyme andthe comparison of its electronic absorption spectra with other variants suggest that the loss in activity isnot due to a conformational change or a defective metal center. Two different reaction mechanisms areproposed and are compared to those of related enzymes. These results also suggest that inhibitors analogousto that reported here may be useful in preventing angiogenesis in cancer and in the treatment of microbialand fungal infections.
-hydroxide)bridges the trigonal bipyramidal (Co1) and octahedral (Co2) cobalt ions. A second solvent (possibly ahydroxide ion) is bound terminally to Co2. A monovalent cation binding site was also identified about 13Å away from the metal center at an interface between the two subdomains of the protein. The first structureof a substrate-like inhibitor, (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L-Leu-L-Val-L-Phe-OMe, bound toa methionine aminopeptidase, has also been determined. This inhibitor coordinates the metal center throughfour interactions as follows: (i) ligation of the N-terminal (3R)-nitrogen to Co2, (ii, iii) bridging coordinationof the (2S)-hydroxyl group, and (iv) terminal ligation to Co1 by the keto oxygen of the pseudo-peptidelinkage. Inhibitor binding occurs with the displacement of two solvent ligands and the expansion of thecoordination sphere of Co1. In addition to the tetradentate, bis-chelate metal coordination, the substrateanalogue forms hydrogen bonds with His79 and His178, two conserved residues within the active site ofall MetAPs. To evaluate their importance in catalysis His79 and His178 were replaced with alanine. Bothsubstitutions, but especially that of His79, reduce activity. The structure of the His79Ala apoenzyme andthe comparison of its electronic absorption spectra with other variants suggest that the loss in activity isnot due to a conformational change or a defective metal center. Two different reaction mechanisms areproposed and are compared to those of related enzymes. These results also suggest that inhibitors analogousto that reported here may be useful in preventing angiogenesis in cancer and in the treatment of microbialand fungal infections.