The molecular basis of the differing kinetic behavior of the two low molecular mass phosphotyrosine protein phosphatase isoforms.
The low molecular mass phosphotyrosine protein phosphatase is a cytosolic enzyme of 18 kDa. Mammalian species contain a single gene that codifies for two distinct isoenzymes; they are produced through alternative splicing and thus differ only in the sequence from residue 40 to residue 73. Isoenzymes differ also in substrate specificity and in the sensitivity to activity modulators. In our study, we mutated a number of residues included in the alternative 40-73 sequence by substituting the residues present in the type 2 isoenzyme with those present in type 1 and subsequently examined the kinetic properties of the purified mutated proteins. The results enabled us to identify the molecular site that determines the kinetic characteristics of each isoform; the residue in position 50 plays the main role in the determination of substrate specificity, while the residues in both positions 49 and 50 are involved in the strong activation of the type 2 low M(r) phosphotyrosine protein phosphatase isoenzyme by purine compounds such as guanosine and cGMP. The sequence 49-50 is included in a loop whose N terminus is linked to the beta 2-strand and whose C terminus is linked to the alpha 2-helix; this loop is very near the active site pocket. Our findings suggest that this loop is involved both in the regulation of the enzyme activity and in the determination of the substrate specificity of the two low M(r) phosphotyrosine protein phosphatase isoenzymes.
Cirri
P
P
Dipartimento di Scienze Biochimiche, Università di Firenze, Italy.
Fiaschi
T
T
Chiarugi
P
P
Camici
G
G
Manao
G
G
Raugei
G
G
Ramponi
G
G
D000595:Amino Acid Sequence; D000818:Animals; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D007527:Isoenzymes; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases