Roles of aspartic acid 181 and serine-222 in intermediate formation and hydrolysis of the mammalian protein-tyrosine-phosphatase PTP1
Roles of aspartic acid 181 and serine-222 in intermediate formation and hydrolysis of the mammalian protein-tyrosine-phosphatase PTP1
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DOI：
10.1021/bi963094r
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发表时间：
1997-04-15
期刊：
影响因子：
2.9
通讯作者：
Dixon, JE
中科院分区：
文献类型：
--
作者：
Lohse, DL;Denu, JM;Dixon, JE
蛋白酪氨酸磷酸酶（PTPases）共享许多保守的氨基酸残基，包括活性位点序列HCXXGXXRS(T)，这与催化作用密切相关。采用定点诱变和动力学分析相结合的方法，研究了哺乳动物PTPase PTP1中两个保守的活性位点残基Asp-181和Ser-222的作用。野生型酶的k(cat)/ k -m和k（cat）的pH谱表明，pK(a)值为5.1和5.44的两个可电离基团必须被去质子化，pK(a)值为4.93的一个基团必须被质子化才能获得最大活性。pK(a)值为5.1的基团为底物磷酸基的二次电离，选择性硫代阴离子失活表明pK(a)值为5.44的残基为C215。在此期间，D181N突变体的ph依赖性谱确定了pK(a)值为4.93的残基为Asp-181，并表明它作为一般的酸性磷酰转移到酶。野生型和D181N突变型酶的快速反应动力学表明，在pH 7.0和30℃时，磷酸酶中间体的形成是限速的，含有S222A突变的酶表现出快速的反应爆发动力学，强烈表明磷酸酶中间体水解是完全限速的，活性位点S222的作用是加速磷酸酶中间体水解的速度。对包含D181N和S222A突变的第三个突变体的动力学分析表明，D181也在磷酸化酶中间体的分解中起一般的碱基作用。
Protein tyrosine phosphatases (PTPases) share a number of conserved amino acid residues, including the active site sequence HCXXGXXRS(T), which are strongly implicated in catalysis. The roles of two conserved active site residues, Asp-181 and Ser-222, were investigated using a combination of site-directed mutagenesis and kinetic analysis in the mammalian PTPase PTP1. The pH profiles for k(cat)/K-m and k(cat) of the wild-type enzyme indicate that two ionizable groups, of pK(a) values 5.1 and 5.44, must be deprotonated and one group with a pK(a) value of 4.93 must be protonated for maximal activity. The group of pK(a) value 5.1 is the second ionization of the substrate phosphate moiety, Selective thiolate anion inactivation indicates the residue with pK(a) value of 5.44 is C215. The pH-dependent profiles of the D181N mutant during establish the residue with pK(a) value of 4.93 to be Asp-181 and suggest that it functions as a general acid phosphoryl transfer to the enzyme. Rapid reaction kinetics of wild-type and D181N mutant enzymes indicate that the formation of the phospho-enzyme intermediate is rate-limiting at pH 7.0 and 30 degrees C. Enzymes containing the S222A mutation exhibited rapid reaction burst kinetics, strongly suggesting that phospho-enzyme intermediate hydrolysis is fully rate-limiting, The role of the active site S222 is to accelerate the rate of phospho-enzyme intermediate hydrolysis. The kinetic analysis of a third mutant, containing both the D181N and S222A mutations, suggests that D181 also serves as a general base in the breakdown of the phospho-enzyme intermediate.