米曲霉GX0011β-果糖基转移酶的化学修饰及活性中心研究

用化学修饰法及其修饰动力学对米曲霉GX0011β-果糖基转移酶的活性中心结构进行了研究。结果表明：NBS、PMSF、EDC能显著抑制酶的活性，底物对这些抑制有明显的保护作用，且残留酶活与修饰剂的浓度相关，抑制均符合拟一级动力学规律，进一步动力学分析，初步认定该酶活性中心包括至少一个丝氨酸（或苏氨酸）、一个色氨酸和一个天冬氨酸(或谷氨酸)残基。pCMB、TNBS能显著抑制酶的活性，但底物对抑制无明显保护作用，推断半胱氨酸和赖氨酸残基可能与维系酶活性中心构象有关，但不是酶活性中心基团。DEPC、AA和NAI对酶的活性抑制作用不明显，排除了组氨酸、精氨酸和酪氨酸残基是该酶活性中心必需基团的可能。

CHEMICAL MODIFICATION AND ACTIVE SITES OF β-FRUCTOSYL-TRANSFERASE FROM Aspergillus oryzae GX0011

TThe active sites of β-fructosyl-transferase from Aspergillus oryzae GX0011 were investigated by means of chemical modification and kinetic study. NBS(N-bromosuccinimide)、PMSF(Phenylmethyl-sulphonyl fluoride)、EDC(Carbodimide) seriously inactivated the enzyme but not in the presence of substrate. The inactivation reactions followed pseudo-first-order kinetics. Further kinetic analysis of the inactivation indicated that at least one serine/threonine residue, one tryptophan residue and one asparic acid/glutamic acid residue might be involved in the active site of the enzyme. PCMB(p-chloromercuribenzoate) and TNBS(2,4,6-trinitrobenzene sulfonate) could inactivate the enzyme even in the presence of substrate. This implied that cysteine and lysine residues might not be in the active site of the enzyme but involved in maintaining the conformation of the enzyme. DEPC(Diethylpyrocarbonate)、AA(acetylacetone) and NAI (N-acetyl-imidazole), did not obviously influence activity, thus eliminating the possibility that histidine, arginine and tyrosine participated in catalysis.