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J M Denu

Publications and source records attributed to J M Denu.

33 records · Page 2Linked to original sources
PubMed1996

Purification and kinetic characterization of the mitogen-activated protein kinase phosphatase rVH6.

The dual specificity protein-tyrosine phosphatase rVH6 belongs to a subfamily of enzymes that have in vivo and in vitro catalytic activity against mitogen-activated protein kinases. A method was developed for the expression and efficient purification of recombinant rVH6 in quantities sufficient for physical and kinetic characterization of the enzyme. Matrix-assisted laser desorption mass spectrometry verified the mass of purified rVH6 to be 43,500 +/- 150, and NH2-terminal sequence analysis confirmed the predicted amino acid sequence. Kinetic characterization of full-length rVH6 identified the critical ionizations involved in the kcat/Km parameter (apparent pKa values 5.1 and 6.6) and revealed a pH-independent kcat value of 0.014 s-1. In an attempt to define the essential catalytic core of this enzyme, amino acids 134-381 of rVH6 were expressed, purified, and characterized enzymatically. Kinetic analysis revealed that the truncated enzyme exhibited a turnover value similar to that of the full-length enzyme (kcat = 0.017 s-1), with p-nitrophenyl phosphate as substrate. Secondary structure prediction and molecular modeling of rVH6 based on the x-ray structure of the dual specificity protein tyrosine phosphatase, VHR, further supported the assignment of residues 134-381 to the core catalytic domain of rVH6. These results demonstrate that the NH2 terminus of rVH6 (residues 1-133) is not required for full enzyme activity and comprises a separate domain that may play a distinct physiological function.

Chromatography, High Pressure Liquid↗
PubMed1996

Transition-state structures for the native dual-specific phosphatase VHR and D92N and S131A mutants. Contributions to the driving force for catalysis.

Isotope effects have been measured for the reaction of the human dual-specific phosphatase VHR with p-nitrophenyl phosphate (pNPP). Isotope effects in the nonbridge oxygen atoms, in the bridge oxygen atom, and in the nitrogen atom were measured by the competitive method using an isotope ratio mass spectrometer. These are isotope effects on V/K, and give information on the chemical step of phosphoryl transfer from substrate to the enzymatic nucleophile Cys-124. With native VHR, 18(V/K)nonbridge = 1.0003 +/- 0.0003, 18(V/K)bridge = 1.0118 +/- 0.0020, and 15(V/K) = 0.9999 +/- 0.0004. The values are similar to the intrinsic isotope effects for the uncatalyzed reaction, indicating that the chemical step is rate-limiting with the pNPP substrate. The transition-state structure resembles that for the uncatalyzed reaction and those previously found for the protein-tyrosine phosphatases YOP51 and PTP1, and is highly dissociative with P-O bond cleavage and protonation of the leaving group by the general acid Asp-92 both well advanced. The D92N mutant exhibits a transition state similar to that of the uncatalyzed reaction of the pNPP dianion, dissociative and with the leaving group departing as the nitrophenolate anion. The S131A mutation causes an increase in the pKa of the nucleophilic Cys, but the isotope effect data are unchanged from those for the native enzyme, indicating no effects of this increase in nucleophilicity on transition-state structure. The double mutant D92N/S131A manifests both the increase in pKa of the nucleophilic Cys and the loss of general acid assistance to the leaving group. In the absence of the general acid, the change in nucleophile pKa results in an increase in 18(V/K)nonbridge from 1.0019 (with D92N) to 1.0031 (with D92N/S131A), indicating loss of P-O nonbridge bond order in the transition state. It is concluded that this is more likely caused by electrostatic effects rather than resulting from increased nucleophile-phosphorus bonding in a less dissociative transition state, although the latter explanation cannot be excluded on the basis of the present data. Electrostatic effects between the thiolate anion nucleophile and the phosphoryl group may be an important part of the driving force for catalysis in this family of enzymes.

Catalysis↗
PubMed1996

Crystal structure of the dual specificity protein phosphatase VHR.

Dual specificity protein phosphatases (DSPs) regulate mitogenic signal transduction and control the cell cycle. Here, the crystal structure of a human DSP, vaccinia H1-related phosphatase (or VHR), was determined at 2.1 angstrom resolution. A shallow active site pocket in VHR allows for the hydrolysis of phosphorylated serine, threonine, or tyrosine protein residues, whereas the deeper active site of protein tyrosine phosphatases (PTPs) restricts substrate specificity to only phosphotyrosine. Positively charged crevices near the active site may explain the enzyme's preference for substrates with two phosphorylated residues. The VHR structure defines a conserved structural scaffold for both DSPs and PTPs. A "recognition region," connecting helix alpha1 to strand beta1, may determine differences in substrate specificity between VHR, the PTPs, and other DSPs.

Amino Acid Sequence↗
PubMed1996

Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis.

Engineering site-specific amino acid substitutions into the protein-tyrosine phosphatase (PTPase) PTP1 and the dual-specific vaccinia H1-related phosphatase (VHR), has kinetically isolated the two chemical steps of the reaction and provided a rare opportunity for examining transition states and directly observing the phosphoenzyme intermediate. Changing serine to alanine in the active-site sequence motif HCXXGXXRS shifted the rate-limiting step from intermediate formation to intermediate hydrolysis. Using phosphorus 31P NMR, the covalent thiol-phosphate intermediate was directly observed during catalytic turnover. The importance of the conserved aspartic acid (D92 in VHR and D181 in PTP1) in both chemical steps was established. Kinetic analysis of D92N and D181N mutants indicated that aspartic acid acts as a general acid by protonating the leaving-group phenolic oxygen. Structure-reactivity experiments with native and aspartate mutant enzymes established that proton transfer is concomitant with P-O cleavage, such that no charge develops on the phenolic oxygen. Steady- and presteady-state kinetics, as well as NMR analysis of the double mutant D92N/S131A (VHR), suggested that the conserved aspartic acid functions as a general base during intermediate hydrolysis. As a general base, aspartate would activate a water molecule to facilitate nucleophilic attack. The amino acids involved in transition-state stabilization for cysteinylphosphate hydrolysis were confirmed by the x-ray structure of the Yersinia PTPase complexed with vanadate, a transition-state mimic that binds covalently to the active-site cysteine. Consistent with the NMR, x-ray, biochemical, and kinetic data, a unifying mechanism for catalysis is proposed.

Amino Acid Sequence↗
PubMed1995

A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase.

Dual-specificity protein-tyrosine phosphatases (dsPTPases) have been implicated in the inactivation of mitogen-activated protein kinases (MAPKs). We have identified a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, except for the substitution of Gly for Cys in the conserved dsPTPase catalytic loop (HCXXGXXR(S/T)). cDNA subcloning and Northern blot analysis in mouse shows poly(A+) hybridization bands of 4.6, 2.4, 1.5, and 1.2 kilobases, with highest abundance in skeletal muscle, testis, and heart. Polymerase chain reaction amplification of reverse-transcribed poly(A+) RNA revealed an alternatively spliced form of STYX containing a unique carboxyl terminus. Bacterially expressed STYX is incapable of hydrolyzing Tyr(P)-containing substrates; however, mutation of Gly120 to Cys (G120C), which structurally mimics the active site of dsPTPases, confers phosphatase activity to this molecule. STYX-G120C mutant hydrolyzes p-nitrophenyl phosphate and dephosphorylates both Tyr(P) and Thr(P) residues of peptide sequences of MAPK homologues. The kinetic parameters of dephosphorylation are similar to human dsPTPase, Vaccinia H1-related, including inhibition by vanadate. We believe this is the first example of a naturally occurring "dominant negative" phosphotyrosine/serine/threonine-binding protein which is structurally related to dsPTPases.

Alternative Splicing↗
PubMed1995

Purification and characterization of the low molecular weight protein tyrosine phosphatase, Stp1, from the fission yeast Schizosaccharomyces pombe.

Genetic screening in fission yeast has identified a gene named stp1+ that rescues cdc25-22 [Mondesert et al. (1994) J. Biol. Chem. 269, 27996-27999]. This gene encodes a 17.4 kDa protein that is 42% identical to members of the low molecular weight protein tyrosine phosphatases (low M(r)PTPases) previously known to exist only in mammalian species. A simple and efficient purification procedure was developed to obtain the homogeneous recombinant yeast low M(r)PTPase, Stp1, in large quantities suitable for kinetic and structural studies. Authentic Stp1 was produced as judged by amino terminal protein sequencing and electrospray ionization mass spectrometry analyses. Stp1 was shown to possess intrinsic phosphatase activity toward both aryl phosphates (such as phosphotyrosine) and alkyl phosphates (such as phosphoserine). Stp1 also dephosphorylated phosphotyrosyl peptide/protein substrates. The yeast enzyme was 6-fold slower than the mammalian enzymes, which made it amenable to pre-steady-state stopped-flow spectroscopic kinetic analysis at 30 degrees C and pH 6.0. Burst kinetics was observed with Stp1 using p-nitrophenyl phosphate as a substrate, suggesting that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate. Interestingly, the bovine heart low M(r)PTPase was capable of removing phosphate groups from both phosphotyrosyl and phosphoseryl/threonyl protein substrates with comparable efficiencies. The low M(r)PTPases, like the Cdc25 family of phosphatases, may represent a new group of dual specificity phosphatases which may be involved in cell cycle control.

Amino Acid Sequence↗
PubMed1995

A catalytic mechanism for the dual-specific phosphatases.

Dual-specific protein-tyrosine phosphatases have the common active-site sequence motif HCXXGXXRS(T). The role of the conserved hydroxyl was investigated by changing serine-131 to an alanine (S131A) in the dual-specific protein-tyrosine phosphatase VHR. The pH profile of the kcat/Km value for the S131A mutant is indistinguishable from that of the native enzyme. In contrast, the kcat value for S131A mutant is 100-fold lower than that for the native enzyme, and the shape of the pH profile was perturbed from bell-shaped in the native enzyme to a pH-independent curve over the pH range 4.5-9.0. This evidence, along with results from a previous study, suggests that the S131A mutation alters the rate-limiting step in the catalytic mechanism. Formation of a phosphoenzyme intermediate appears to be rate-limiting with the native enzyme, whereas in the S131A mutant breakdown of the intermediate is rate-limiting. This was confirmed by the appearance of a burst of p-nitrophenol formation when p-nitrophenyl phosphate rapidly reacted with the S131A enzyme in a stopped-flow spectrophotometer. Loss of this hydroxyl group at the active site dramatically diminished the ability of the enzyme to hydrolyze the thiol-phosphate intermediate without exerting any significant change in the steps leading to and including the formation of the intermediate. Consistent with rate-limiting intermediate formation in the native enzyme, the rate of burst in the S131A mutant was 1.5 s-1, which agrees well with the kcat value of 5 s-1 observed for native enzyme. The amplitude of the burst was stoichiometric with final enzyme concentration, and the slow linear rate (0.06 s-1) of p-nitrophenol formation after the burst was in agreement with the steady-state determined value of kcat (0.055 s-1).

Alanine↗
PubMed1995

The catalytic role of aspartic acid-92 in a human dual-specific protein-tyrosine-phosphatase.

The mechanism of catalysis for the human dual-specific (vaccinia H1-related) protein-tyrosine-phosphatase was investigated. The pH dependence of the kcat value is bell-shaped when p-nitrophenyl phosphate was employed as a model substrate. The kcat/Km pH profile rises with a slope of 2 and decreases with a slope of -1, indicating that two groups must be unprotonated and one group must be protonated for activity. An amino acid residue with an apparent pKa value of 5.5 +/- 0.2 must be unprotonated and a residue with a pKa value of 5.7 must be unprotonated for activity. The pKa value of the catalytic cysteine-124 (C124) was 5.6 +/- 0.1. The aspartic acid-92-asparagine (D92N) mutant enzyme was 100-fold less active than the native enzyme and exhibited the loss of the basic limb in the pH profiles, suggesting that in the native enzyme D92 must be protonated for activity. The D92 residue is conserved throughout the entire family of dual-specific phosphatases. Mutants glutamic acid-6-glutamine, glutamic acid-32-glutamine, aspartic acid-14-asparagine, and aspartic acid-110-asparagine had less than a 2-fold effect on the kinetic parameters when compared to native enzyme. Based upon the lack of a "burst" in rapid reaction kinetics, formation of the intermediate is rate-limiting with both native and D92N mutant enzymes. In agreement with rate-limiting formation of the intermediate, the pKa value of 5.5 for the group which must be unprotonated for activity was assigned to C124.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

The purification and characterization of a human dual-specific protein tyrosine phosphatase.

An expression and purification method was developed to obtain the recombinant human dual-specific protein tyrosine phosphatase (PTPase) VHR in quantities suitable for both kinetic studies and crystallization. Physical characterization of the homogeneous recombinant protein verified the mass to be 20,500 +/- 100 by matrix-assisted laser desorption mass spectrometry, confirmed the anticipated NH2-terminal amino acid sequence and demonstrated that the protein exists as a monomer. Conditions were developed to obtain crystals which were suitable for x-ray structure determination. Using synthetic diphosphorylated peptides corresponding to MAP177-189 (mitogen-activated protein) kinase (DHTG-FLpTEpYVATR), an assay was devised which permitted the determination of the rate constants for dephosphorylation of the diphosphorylated peptide on threonine and tyrosine residues. The diphosphorylated peptides are preferred over the singly phosphorylated on tyrosine by 3-8-fold. The apparent second-order rate constant kcat/Km for dephosphorylation of phosphotyrosine on DHTGFLpTEpYVATR was 32,000 M-1 S-1 while dephosphorylation of phosphothreonine was 14 M-1 S-1 (pH 6). The reaction of DHTGFLpTEpYVATR with VHR is ordered, with rapid dephosphorylation on tyrosine occurring first followed by slow dephosphorylation on threonine. Similar results were obtained with F(NLe)(N-Le)pTPpYVVTR, a peptide corresponding to a MAP kinase-like protein (JNK1(180-189)) which is involved in the stress response signaling pathway.

Amino Acid Sequence↗
PubMed1994

The catalytic role of Cys124 in the dual specificity phosphatase VHR.

The recombinant human Vaccinia virus H1-related protein tyrosine phosphatase, (VHR PTPase) possesses intrinsic Tyr and Thr/Ser phosphatase activities. Both activities were abolished by a single amino acid substitution, C124S. When VHR was incubated with a 32P-labeled phosphotyrosine-containing substrate and then rapidly denatured, enzyme-associated 32P was evident following SDS-polyacrylamide gel electrophoresis. The formation of 32P-labeled protein could be blocked in the presence of an unlabeled substrate. VHR-associated 32P was sensitive to iodine but insensitive to pyridine and hydroxylamine. The catalytically inactive C124S mutant would not form a 32P-labeled enzyme. Furthermore, VHR phosphatase could be selectively inactivated by the alkylating agent iodoacetate. The inactivation resulted from the specific covalent modification of Cys124. Collectively these results suggest that a thiol-phosphate enzyme intermediate is formed when Cys124 of VHR accepts a phosphate from the substrate. Our results also demonstrate that the dual specificity phosphatases and the tyrosine-specific PTPases employ similar catalytic mechanisms.

Amino Acid Sequence↗
PubMed1994

pH and kinetic isotope effects on the oxidative half-reaction of D-amino-acid oxidase.

D-Amino-acid oxidase catalyzes the oxidation of D-amino acids to imino acids. In the oxidative half-reaction, oxygen reacts with the reduced enzyme-imino acid complex to reoxidize the bound FAD. This is then followed by dissociation of the imino acid. The effects of pH and D2O on the kinetics of the oxidative half-reaction of D-amino-acid oxidase have been determined with glycine, D-alanine, and D-serine as substrates. Reaction of the reduced enzyme with oxygen requires that a group with a pKa value of about 10.5 be protonated and a group with a pKa value of 8.5 be deprotonated. The former value is not seen with D-alanine as substrate; the latter is only seen with glycine. No solvent isotope effects are seen on the V/KO2 value with D-alanine, consistent with rate-limiting electron transfer. Product release involves a pH-dependent conformational change. This is rate-limiting at all pH values with D-alanine as substrate. Significant solvent isotope effects are seen on the Vmax value with D-alanine. The proton inventory at high pH is linear, consistent with release of a single proton in the slow step; at pH 6 the solvent inventory is bowl-shaped, consistent with a solvent isotope effect on the conformation of the protein. With glycine the DV value increases to the intrinsic value at pH 10.5; this establishes that CH bond cleavage becomes rate-limiting with this substrate above pH 10.

Alanine↗
PubMed1994

Intrinsic primary, secondary, and solvent kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase: evidence against a concerted mechanism.

D-Amino acid oxidase catalyzes the oxidation of D-amino acids to imino acids with subsequent transfer of the electrons to molecular oxygen. Proposed mechanisms for the mode of cleavage of the substrate CH bond include stepwise formation of a carbanion, followed by attack of the carbanion on the enzyme-bound FAD, direct hydride transfer of the substrate alpha-hydrogen to the FAD, and transfer of a hydride from the substrate amino group to the FAD. Conditions have previously been established under which large, limiting, primary deuterium kinetic isotope effects can be measured with D-alanine, D-serine, and glycine as substrates for D-amino acid oxidase [Denu, J. M., & Fitzpatrick, P. F. (1992) Biochemistry 31, 8207-8215]. To determine whether these values are the intrinsic isotope effects, primary tritium kinetic isotope effects have been determined with these three substrates. The values are 12.6, 8.6, and 6.4, respectively. These values are consistent with expression of the intrinsic isotope effects under these conditions, allowing for determination of the values of the intrinsic deuterium effects as 5.7, 4.5, and 3.6 for D-alanine, D-serine, and glycine, respectively. Under these conditions, the alpha-secondary tritium kinetic isotope effect with glycine, the beta-secondary deuterium kinetic isotope effect with D-alanine, and the solvent kinetic isotope effect with D-serine are all indistinguishable from unity. These results are not consistent with concerted mechanisms for CH bond cleavage with this enzyme, but are fully consistent with the involvement of a carbanion intermediate.

Alanine↗
PubMed1992

pH and kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase.

Primary deuterium kinetic isotope and pH effects on the reduction of D-amino acid oxidase by amino acid substrates were determined using steady-state and rapid reaction methods. With D-serine as substrate, reduction of the enzyme-bound FAD requires that a group with a pKa value of 8.7 be unprotonated and that a group with a pKa value of 10.7 be protonated. The DV/Kser value of 4.5 is pH-independent, establishing that these pKa values are intrinsic. The limiting rate of reduction of the enzyme shows a kinetic isotope effect of 4.75, consistent with this as the intrinsic value. At high enzyme concentration (approximately 15 microM) at pH 9,D-serine is slightly sticky (k3/k2 = 0.8), consistent with a decrease in the rate of substrate dissociation. With D-alanine as substrate, the pKa values are perturbed to 8.1 and 11.5. The DV/Kala value increases from 1.3 at pH 9.5 to 5.1 at pH 4, establishing that D-alanine is sticky with a forward commitment of approximately 10. The effect of pH on the DV/Kala value is consistent with a model in which exchange with solvent of the proton from the group with pKa 8.7 is hindered and is catalyzed by H2O and OH- above pH 7 and by H3O+ and H2O below pH 7. With glycine, the pH optimum is shifted to a more basic value, 10.3. The DV/Kgly value increases from 1.26 at pH 6.5 to 3.1 at pH 10.7, consistent with fully reversible CH bond cleavage followed by a pH-dependent step. At pH 10.5, the kinetic isotope effect on the limiting rate of reduction is 3.4.

Alanine↗