INTERCONVERSION OF THE KINETIC IDENTITIES OF THE TANDEM CATALYTIC DOMAINS OF RECEPTOR-LIKE PROTEIN-TYROSINE-PHOSPHATASE PTP-ALPHA BY 2 POINT MUTATIONS IS SYNERGISTIC AND SUBSTRATE-DEPENDENT

The two tandem homologous catalytic domains of PTP alpha possess diffe rent kinetic properties, with the membrane proximal domain (D1) exhibi ting much higher activity than the membrane distal (D2) domain. Sequen ce alignment of PTP alpha-D1 and -D2 with the D1 domains of other rece ptor-like PTPs, and modeling of the PTP alpha-D1 and -D2 structures, i dentified two nonconserved amino acids in PTP alpha-D2 that may accoun t for its low activity. Mutation of each residue (Val-536 or Glu-671) to conform to its invariant counterpart in PTP alpha-D1 positively aff ected the catalytic efficiency of PTP alpha-D2 toward the in vitro sub strates para-nitrophenylphosphate and the phosphotyrosyl-peptide RR-sr c. Together, they synergistically transformed PTP alpha-D2 into a phos phatase with catalytic efficiency for para-nitrophenylphosphate equal to PTP alpha-D1 but not approaching that of PTP alpha-D1 for the more complex substrate RR-src, In vivo, no gain in D2 activity toward p59(f yn) was effected by the double mutation. Alteration of the two corresp onding invariant residues in PTPa-D1 to those in D2 conferred Da-like kinetics toward all substrates, Thus, these two amino acids are critic al for interaction with phosphotyrosine but not sufficient to supply P TP alpha-D2 with a D1-like substrate specificity for elements of the p hosphotyrosine microenvironment present in RR-src and p59(fyn). Whethe r the structural features of D2 can uniquely accommodate a specific ph osphoprotein substrate or whether D2 has an alternate function in PTP alpha remains an open question.