The kinetic and structural basis for substrate specificity of the low molecular weight phosphotyrosyl phosphatase

Authors
Publisher
Purdue University
Publication Date
Keywords
• Chemistry
• Biochemistry
Disciplines
• Biology
• Mathematics

Abstract

The low M$\sb{\rm r}$ PTPases are a family of 18 kDa proteins that are found in vertebrates, yeast and bacteria. BPTP and the human isoenzymes (HCPTP-A and HCPTP-B) are prototypical members of this enzyme family. Structural data for BPTP suggests that several conserved residues are directly involved in the formation of the active site. Site directed mutagenesis, steady state kinetics, and protein stability studies showed that Asn-15, His-72, and Ser-19 served structural functions that allowed the active site to adopt the optimal geometry for phosphate binding. Leaving group dependence studies, partitioning experiments, and pH dependence studies also showed that Ser-19 was important for facilitating ionization of the nucleophilic cysteine and maintaining it in an optimal position for nucleophilic attack. The X-ray crystallographic structures of BPTP and HCPTP-A revealed several residues that may play important role(s) in determining the substrate specificity of the A- and B-isoenzymes. These included residues Trp49, Asn-50 and Arg-53 in HCPTP-B, and Tyr-49, Glu-50 and Asn-53 in HCPTP-A. Site directed mutagenesis, steady state kinetics, effector studies, and time dependent dephosphorylation studies showed that although residues 49 and 50 were important determinants of substrate specificity with small pseudosubstrates, their effects were not sufficient to explain differences in substrate specificity with physiologically-based peptides. Site directed mutagenesis was also used in order to elucidate the roles of several conserved residues in determining the specificity of BPTP for aryl substrates. Steady state kinetics suggested that Tyr-131 and Tyr-132 were important for stacking against the tyrosyl portion of the substrate as well as providing selectivity against phosphoseryl and phosphothreonyl substrates. Steady state kinetics using sterically hindered substrates also revealed that Ile-16 plays a major role in determining the sensitivity of the enzyme to steric factors. ^

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