Abstract

Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H⁴⁹²EXXH⁴⁹⁶. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pK_a (pK_e1), while no residue was assigned to contribute alkaline pK_a (pK_e2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N³-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH₂ to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pK_e2.

中文翻译：

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组氨酸和酪氨酸残基在霍乱弧菌的胶原酶活性位点中的作用。

摘要

来自霍里森氏菌（Grimontia hollisae）菌株1706B（Ghcol）的胶原酶是具有锌结合基序H ⁴⁹² EXXH ⁴⁹⁶的锌金属蛋白酶。它具有比来自组织溶梭菌的胶原酶更高的胶原降解活性，后者在工业上被广泛使用。我们之前检查了pH和温度对Ghcol活性的依赖性。Glu493被认为对酸性p K _a（p K _e1）有贡献，而没有残基对碱性p K _a（p K _e2）有贡献。）。在这项研究中，我们在活动位点及其附近的His或Tyr残基处引入了9个单突变。我们的结果表明，H412A，H485A，Y497A，H578A和H737A保留了水解胶原蛋白和明胶的活性，而H426A，H492A，H496A和Y568A则缺乏它们。活性变体H412A，H485A，H578A和H737A以及非活性变体H492A和H496A的纯化成功。H412A优选的（7-甲氧基香豆素-4-基）乙酰基-L-Lys-L-Pro-L-Leu-Gly-L-Leu- [ N ^3-（2,4-二硝基苯基）-L-2,3-二氨基丙酰基] -L-丙氨酸-L-精氨酸-NH ₂H485A比胶原蛋白更适合于胶原蛋白，这表明His412和His485对于底物特异性很重要。活性变体Y497A和非活性变体H426A和Y568A的纯化均未成功，表明这三个残基对稳定性很重要。根据报道的梭菌胶原酶的晶体结构，Ghcol的Tyr568被建议参与催化，可能是p K _e2的可电离残基。