Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis

Previous enzyme kinetic and structural studies have revealed a critical role for Asp¹⁸¹ (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp¹⁸¹ functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp ¹⁸¹ is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPα and PTPε. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His¹⁸² in PTP1B and Phe¹⁸² in PTPH1, we demonstrate here that His¹⁸²-PTPs, in comparison with Phe ¹⁸²-PTPs, have significantly decreased k_cat values, and to a lesser degree, decreased k_cat/K_m values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His¹⁸² is due to interactions with Asp ¹⁸¹ and with Gln²⁶². We conclude that residue 182 can modulate the functionality of both Asp¹⁸¹ and Gln²⁶² and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.