Jeffcoat R, Kirkwood S
J Biol Chem. 1987 Jan 25;262(3):1088-91.
The enzyme, exo-beta-(1-3)-D-glucanase, (EC 3.2.1-) obtained from a culture filtrate of Basidiomycete sp. QM 806, has been obtained in a highly purified form and preliminary investigations on its mechanism of action have been reported (Peterson, D. R., and Kirkwood, S. (1975) Carbohydr. Res. 41, 273-283). Studies reported in this paper, have provided strong evidence for the role of histidine in the catalytic site of this carbohydrase. Chemical modifications of the amino acid residues in the enzyme with diazotized 5-amino-1H-tetrazole or tetranitromethane caused irreversible loss of enzyme activity which varied according to the time of exposure to, or concentration of the inhibitor. Prior incubation of the enzyme with a substrate considerably reduced the extent of this inhibition. Amino acid analysis of the enzyme treated in these ways clearly indicated that the substrate protected histidine residues from chemical modification by the diazotized 5-amino-1H-tetrazole. Chemical modification of both histidine and tyrosine residues were effected by incubating the enzyme with the inhibitors described above. Although evidence is presented to suggest that tyrosine is not directly involved in the active site of the enzyme (the catalytic site or the binding site), the role of this residue in the maintenance of the enzyme conformation is discussed. Enzyme assays carried out either in aqueous or deuterated buffer systems provided further evidence which is consistent with the proposed enzyme mechanism.
从担子菌QM 806的培养滤液中获得的外-β-(1-3)-D-葡聚糖酶（EC 3.2.1-）已被高度纯化，并已有关于其作用机制的初步研究报道（彼得森，D.R.，和柯克伍德，S.（1975年）《碳水化合物研究》41卷，273 - 283页）。本文所报道的研究为组氨酸在这种碳水化合物酶催化位点中的作用提供了有力证据。用重氮化的5-氨基-1H-四唑或四硝基甲烷对该酶中的氨基酸残基进行化学修饰，导致酶活性不可逆丧失，其丧失程度随暴露于抑制剂的时间或抑制剂浓度而变化。酶与底物预先温育可显著降低这种抑制程度。对以这些方式处理过的酶进行氨基酸分析清楚地表明，底物可保护组氨酸残基不被重氮化的5-氨基-1H-四唑化学修饰。通过将酶与上述抑制剂一起温育实现了对组氨酸和酪氨酸残基的化学修饰。尽管有证据表明酪氨酸不直接参与酶的活性位点（催化位点或结合位点），但本文讨论了该残基在维持酶构象中的作用。在水性或氘代缓冲体系中进行的酶活性测定提供了与所提出的酶作用机制相一致的进一步证据。
