Substrate trapping in cysteine to serine mutated PTPases

SUBSTRATE TRAPPING IN CYSTEINE TO SERINE MUTATED PTPases 

The cysteine residue in the catalytic loop (Cysl2 in LMPTP, Cys215 in PTPIB) is the essential nucleophile for PTPase activity. Experiments show that cysteine to serine mutants are completely inactive but can still bind substrate molecules. The ability to bind substrates without hydrolyzing them is called substrate trapping and has been exploited when searching for native PTPase 

The simulations were performed using the PTPase crystal structures as above. The PTPIB structure was a serine mutant with a phosphotyrosine ligand, which was manually replaced by phenyl phosphate. Acidic and basic residues close to the active site were charged whereas those outside the simulation sphere and distant to the active site were replaced by polar neutral groups giving the system a total charge of zero [37]. 

Each simulation was prepared by slow heating of the system from 1 to 300 K followed by 100 ps equilibration at 300 K. The perturbations were performed at 

The results shown in Table 1 show that the simulations forwards and backwards yield similar energies with small standard deviations. The negative AAG,i a values predict that the serine mutants of both LMPTP and PTPIB bind phenyl phosphate 7-8 kcal/mol more strongly than the native enzymes. The stronger stabilization of the enzyme-substrate complex relative to the transition state in addition to the higher pAT of the hydroxyl group compared to the thiol group are therefore likely to be the major reasons for the complete lack of activity. 

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