The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae
The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae
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DOI：
10.1093/jb/mvaa055
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发表时间：
2020-10-01
影响因子：
2.7
通讯作者：
Yasukawa, Kiyoshi
中科院分区：
文献类型：
--
作者：
Hayashi, Kaichi;Ikeuchi, Takeaki;Yasukawa, Kiyoshi
来自Grimontia hollisae菌株1706 B（Ghcol）的胶原酶是具有锌结合基序（HEXXH 496）-E-492的锌金属蛋白酶。它具有比工业上广泛使用的溶组织梭菌胶原酶更高的胶原降解活性。我们先前检查了Ghcol活性的pH和温度依赖性; Glu 493被认为贡献酸性pK（a）（pK（e1）），而没有残基被分配贡献碱性pK（a）（pK（e2））。在这项研究中，我们引入了9个单突变的His或Tyr残基和附近的活性位点。结果表明，H412 A、H485 A、Y 497 A、H578 A和H737 A具有水解胶原和明胶的活性，而H426 A、H492 A、H496 A和Y 568 A缺乏水解胶原和明胶的活性。活性变体H412 A、H485 A、H578 A和H737 A沿着无活性变体H492 A和H496 A的纯化是成功的。H412 A优选（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-Ala-L-Arg-NH 2而不是胶原，而H485 A优选胶原而不是肽，这表明His 412和His 485对于底物特异性是重要的。活性变体Y 497 A和非活性变体H426 A和Y 568 A的纯化不成功，表明这三个残基对于稳定性是重要的。基于已报道的梭菌胶原酶的晶体结构，Ghcol的Tyr 568被认为参与催化作用，并且可能是pK（e2）的可电离残基。
Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif (HEXXH496)-E-492. 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-[N-3-(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).