The role of Cys-17 in the pyridoxal 5'-phosphate inhibition of the bovine liver low M(r) phosphotyrosine protein phosphatase.
Mammalian tissues contain two low M(r) phosphotyrosine protein phosphatase isoforms (type-1 and type-2) that differ in the 40-73 amino-acid sequence. Only one isoform (type-2) is strongly inhibited by pyridoxal 5'-phosphate, whereas the other is poorly inhibited by this compound. The mechanism of pyridoxal 5'-phosphate inhibition of the bovine liver enzyme (a type-2 isoform) has been studied by kinetic methods using a series of pyridoxal 5'-phosphate analogues. These studies indicate that pyridoxal 5'-phosphate interacts with the enzyme in both the phosphate and aldehyde groups. Active site-directed mutagenesis has been used to investigate the sites of pyridoxal 5'-phosphate binding. Our results indicate that Cys-17, essential for enzyme activity, interacts with the phosphate moiety of pyridoxal 5'-phosphate. On the other hand, Cys-12, which is also involved in the catalytic mechanism, does not participate in pyridoxal 5'-phosphate binding.
Cirri
P
P
Department of Biochemical Sciences, University of Florence, Firenze, Italy.
Chiarugi
P
P
Camici
G
G
Manao
G
G
Pazzagli
L
L
Caselli
A
A
Barghini
I
I
Cappugi
G
G
Raugei
G
G
Ramponi
G
G
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D019000:Phosphotyrosine; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics; D012545:Schiff Bases; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000494:pharmacology
D012545:Schiff Bases; D019000:Phosphotyrosine; D014443:Tyrosine; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine