Human γ-Glutamyl Transpeptidase Mutants Involving Conserved Aspartate Residues and the Unique Cysteine Residue of the Light Subunit (*)
Human γ-Glutamyl Transpeptidase Mutants Involving Conserved Aspartate Residues and the Unique Cysteine Residue of the Light Subunit (*)
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涉及保守天冬氨酸残基和轻亚基独特半胱氨酸残基的人γ-谷氨酰转肽酶突变体（*）
DOI：
--
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发表时间：
1995
影响因子：
4.8
通讯作者：
A. Meister
中科院分区：
文献类型：
--
作者：
Y. Ikeda;J. Fujii;Naoyuki Taniguchi;A. Meister
制备了在几种物种中保守的Asp残基轻亚基上和独特的半胱氨酸残基（Cys-454）处具有氨基酸取代的突变的人γ-谷氨酰转肽酶，并在杆状病毒昆虫细胞系统中表达。用Ala或Glu取代Asp-423导致酶活性的主要损失，这与Asp-423对活性至关重要的结论一致。其中Cys-454被Ala取代的突变体是完全活性的，这表明独特的轻亚基硫醇不需要催化。对L-γ-谷氨酰-对硝基苯胺水解反应动力学的分析表明，Asp-423突变体的活性下降是由于其底物Km值极高，比野生型酶高1000倍以上，而Vmax仅下降不到90倍。结果表明，Asp-423和较小程度的Asp-422与γ-谷氨酰供体底物的α-氨基发生静电相互作用。虽然需要进一步研究来评估反应涉及电荷（或质子）中继系统功能的可能性，但目前的工作表明底物的γ-谷氨酰部分与酶上的特定基团静电结合;这有助于γ-谷氨酰酶的形成。
Mutant human γ-glutamyl transpeptidases with amino acid substitutions on the light subunit at the Asp residues conserved among several species, and at the unique cysteine residue (Cys-454), were prepared and expressed in a baculovirus insect cell system. Replacement of Asp-423 by Ala or Glu led to major loss of enzyme activity, consistent with the conclusion that Asp-423 is essential for activity. A mutant in which Cys-454 was replaced by Ala was fully active, indicating that the unique light subunit thiol is not required for catalysis. Kinetic analysis of the hydrolysis reaction of L-γ-glutamyl-p-nitroanilide indicated that the decreased activity of Asp-423 mutants is the consequence of an extremely high substrate Km value, which is more than a 1000-fold greater than that for the wild-type enzyme, whereas the Vmax is decreased only less than 90-fold. The results suggest that Asp-423, and to a lesser extent Asp-422, interact electrostatically with the α-amino group of the γ-glutamyl donor substrate. Although further studies are required to evaluate the possibility that the reaction involves function of a charge (or proton) relay system, the present work suggests that the γ-glutamyl moiety of the substrate binds electrostatically to specific groups on the enzyme; this facilitates γ-glutamyl enzyme formation.
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人γ-谷氨酰转肽酶的克隆和核苷酸序列。
DOI：
10.1073/pnas.85.23.8840
发表时间：
1988
影响因子：
11.1
作者：
Rajpert-DeMeyts,E;Heisterkamp,N;Groffen,J
通讯作者：
Groffen,J
DOI：
--
发表时间：
1994-08
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
E. Stole;T. Smith;J. Manning;A. Meister
通讯作者：
E. Stole;T. Smith;J. Manning;A. Meister
γ-谷氨酰转肽酶活性位点残基的化学修饰。
DOI：
--
发表时间：
1995
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Smith,TK;Meister,A
通讯作者：
Meister,A
影响因子：
56.9
作者：
CRAIK, CS;ROCZNIAK, S;RUTTER, WJ
通讯作者：
RUTTER, WJ
γ-谷氨酰转肽酶与谷胱甘肽的相互作用涉及重亚基的特定精氨酸和赖氨酸残基。
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Stole,E;Meister,A
通讯作者：
Meister,A