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A missense methionine mutation augments catalytic activity but reduces thermal stability in two protein tyrosine phosphatases.

ABSTRACT: Recent data sets that catalog the missense mutations in thousands of human genomes have revealed a vast and largely unexplored world of non-canonical protein sequences that are expressed in humans. The functional consequences of most human missense mutations, however, are unknown, and the accuracy with which their effects can be predicted by computational algorithms remains unclear. Among humans of European descent, the most common missense mutation in the catalytic domain of SH2-containing protein tyrosine phosphatase 1 (SHP-1) converts the enzyme's canonical valine 451 to methionine (V451M). The V451M mutation lies in a conserved motif adjacent to the protein tyrosine phosphatase (PTP) consensus sequence and is predicted to compromise catalytic function. In this study it is shown that, counter to prediction, V451M SHP-1 possesses increased catalytic activity as compared to the wild-type enzyme. Additionally, a PTP-wide search of missense-mutation data revealed a variant of one other PTP, Fas-associated PTP (FAP-1), that contains a methionine mutation at the position corresponding to 451 of SHP-1 (T2406M FAP-1). It is shown here that the T2406M mutation increases FAP-1's PTP activity, to a degree that is comparable to the activation deriving from the V451M mutation in SHP-1. Although the two non-canonical methionine residues confer increased activity at moderate temperatures, both V451M SHP-1 and T2406M FAP-1 are less thermally stable than their canonical counterparts, as demonstrated by the mutants' strongly reduced activities at high temperatures. These results highlight the challenges in predicting the functional consequences of missense mutations, which can differ under varying conditions, and suggest that, with regard to position 451/2406, canonical PTP domains have "chosen" stability over optimized activity during the course of evolution.

SUBMITTER: Bishop AC

PROVIDER: S-EPMC5118098 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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A missense methionine mutation augments catalytic activity but reduces thermal stability in two protein tyrosine phosphatases.

Bishop Anthony C AC

Biochemical and biophysical research communications 20161102 1-2

Recent data sets that catalog the missense mutations in thousands of human genomes have revealed a vast and largely unexplored world of non-canonical protein sequences that are expressed in humans. The functional consequences of most human missense mutations, however, are unknown, and the accuracy with which their effects can be predicted by computational algorithms remains unclear. Among humans of European descent, the most common missense mutation in the catalytic domain of SH2-containing prot ...[more]

PMID: 27816449

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Project description:Protein tyrosine phosphatases (PTPs) play an important role in the regulation of mammalian signal transduction. During some cell signaling processes, the generation of endogenous hydrogen peroxide inactivates selected PTPs via oxidation of the enzyme's catalytic cysteine thiolate group. Importantly, low-molecular weight and protein thiols in the cell have the potential to regenerate the catalytically active PTPs. Here we examined the recovery of catalytic activity from two oxidatively inactivated PTPs (PTP1B and SHP-2) by various low-molecular weight thiols and the enzyme thioredoxin. All monothiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rate constants that varied by a factor of approximately 8. In general, molecules bearing low-pKa thiol groups were particularly effective. The biological thiol glutathione repaired oxidized PTP1B with an apparent second-order rate constant of 0.023 ± 0.004 M(-1) s(-1), while the dithiol dithiothreitol (DTT) displayed an apparent second-order rate constant of 0.325 ± 0.007 M(-1) s(-1). The enzyme thioredoxin regenerated the catalytic activity of oxidized PTP1B at a substantially faster rate than DTT. Thioredoxin (2 ?M) converted oxidized PTP1B to the active form with an observed rate constant of 1.4 × 10(-3) s(-1). The rates at which these agents regenerated oxidized PTP1B followed the order Trx > DTT > GSHand comparable values observed at 2 ?M Trx, 4 mM DTT, and 60 mM GSH. Various disulfides that are byproducts of the reactivation process did not inactivate native PTP1B at concentrations of 1-20 mM. The common biochemical reducing agent tris(2-carboxyethyl)phosphine regenerates enzymatic activity from oxidized PTP1B somewhat faster than the thiol-based reagents, with a rate constant of 1.5 ± 0.5 M(-1) s(-1). We observed profound kinetic differences between the thiol-dependent regeneration of activity from oxidized PTP1B and SHP-2, highlighting the potential for structural differences in various oxidized PTPs to play a significant role in the rates at which low-molecular weight thiols and thiol-containing enzymes such as thioredoxin and glutaredoxin return catalytic activity to these enzymes during cell signaling events.

Project description:The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >10(5)-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >10(5)-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3-18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A cocrystal structure of PTP1B bound with a nephrin pY(1193) peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities, and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic activities with respect to PTP in vivo substrate specificity and biological functions are discussed.

Dataset Information

A missense methionine mutation augments catalytic activity but reduces thermal stability in two protein tyrosine phosphatases.

Publications

A missense methionine mutation augments catalytic activity but reduces thermal stability in two protein tyrosine phosphatases.

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