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首页> 外文期刊>The FEBS journal >Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris
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Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris

机译：对寻常嗜热放线菌的羧肽酶T广泛的底物特异性的结构见解

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摘要

The crystal structures of carboxypeptidase T (CpT) complexes with phenylalanine and arginine substrate analogs - benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid - were determined by the molecular replacement method at resolutions of 1.57 angstrom and 1.62 angstrom to clarify the broad substrate specificity profile of the enzyme. The conservative Leu211 and Leu254 residues (also present in both carboxypeptidase A and carboxypeptidase B) were shown to be structural determinants for recognition of hydrophobic substrates, whereas Asp263 was for recognition of positively charged substrates. Mutations of these determinants modify the substrate profile: the CpT variant Leu211Gln acquires carboxypeptidase B-like properties, and the CpT variant Asp263Asn the carboxypeptidase A-like selectivity. The Pro248-Asp258 loop interacting with Leu254 and Tyr255 was shown to be responsible for recognition of the substrate's C-terminal residue. Substrate binding at the S1 subsite leads to the ligand-dependent shift of this loop, and Leu254 side chain movement induces the conformation rearrangement of the Glu277 residue crucial for catalysis. This is a novel insight into the substrate selectivity of metallocarboxypeptidases that demonstrates the importance of interactions between the S1 subsite and the catalytic center.
机译：通过分子置换法以1.57埃和1.62埃的分辨率确定了羧肽酶T（CpT）与苯丙氨酸和精氨酸底物类似物-苄基琥珀酸和（2-胍基乙基巯基）琥珀酸的晶体结构，以阐明广泛的底物特异性酶。已显示保守的Leu211和Leu254残基（也存在于羧肽酶A和羧肽酶B中）是识别疏水性底物的结构决定因素，而Asp263用于识别带正电的底物。这些决定因素的突变修饰了底物谱：CpT变体Leu211Gln获得了羧肽酶B样特性，而CpT变体Asp263Asn则获得了羧肽酶A样选择性。 Pro248-Asp258环与Leu254和Tyr255相互作用被证明负责识别底物的C末端残基。底物在S1子位点的结合导致该环的配体依赖性移动，Leu254侧链运动诱导了对催化至关重要的Glu277残基的构象重排。这是对金属羧肽酶底物选择性的新颖见解，证明了S1亚位点与催化中心之间相互作用的重要性。

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