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The biosynthesis of prostate specific antigen (PSA) was studied in human epidermoid carcinoma, (KB) cells and in normal human embryonic lung (WI-38)cells. The prostate carcinoma cell line, PC-35F12 was used as a control. PSA specific antibodies were used to precipitate the immunologically reactive peptides from cell extracts and conditioned media. The immunoprecipitates were analyzed by electrophoresis followed by fluorgraphy. Human PSA is initially synthesized as 32-kDa secretory glycopeptide containing one N-linked oligosaccharide and then processed to a 34-kDa secretory glycoprotein in KB cells. PSA is also expressed in normal human embryonic lung cells, WI-38. These results confirm that PSA expression is not prostate specific, but is also found in some nonprostatic cells. From these results, we conclude that PSA may play an important physiologic role in several tissues.

Low-molecular weight protein tyrosine phosphatases are virtually ubiquitous, which implies that they have important cellular functions. We present here the 2.2 A resolution X-ray crystallographic structure of wild-type LTP1, a low-molecular weight protein tyrosine phosphatase from Saccharomyces cerevisiae. We also present the structure of an inactive mutant substrate complex of LTP1 with p-nitrophenyl phosphate (pNPP) at a resolution of 1.7 A. The crystal structures of the wild-type protein and of the inactive mutant both have two molecules per asymmetric unit. The wild-type protein crystal was grown in HEPES buffer, a sulfonate anion that resembles the phosphate substrate, and a HEPES molecule was found with nearly full occupancy in the active site. Although the fold of LTP1 resembles that of its bovine counterpart BPTP, there are significant changes around the active site that explain differences in their kinetic behavior. In the crystal of the inactive mutant of LTP1, one molecule has a pNPP in the active site, while the other has a phosphate ion. The aromatic residues lining the walls of the active site cavity exhibit large relative movements between the two molecules. The phosphate groups present in the structures of the mutant protein bind more deeply in the active site (that is, closer to the position of nucleophilic cysteine side chain) than does the sulfonate group of the HEPES molecule in the wild-type structure. This further confirms the important role of the phosphate-binding loop in stabilizing the deep binding position of the phosphate group, thus helping to bring the phosphate close to the thiolate anion of nucleophilic cysteine, and facilitating the formation of the phosphoenzyme intermediate.

Although the activation of low-molecular weight protein tyrosine phosphatases by certain purines and purine derivatives was first described three decades ago, the mechanism of this rate enhancement was unknown. As an example, adenine activates the yeast low-molecular weight protein tyrosine phosphatase LTP1 more than 30-fold. To examine the structural and mechanistic basis of this phenomenon, we have determined the crystal structure of yeast LTP1 complexed with adenine. In the crystal structure, an adenine molecule is found bound in the active site cavity, sandwiched between the side chains of two large hydrophobic residues at the active site. Hydrogen bonding to the side chains of other active site residues, as well as some water-mediated hydrogen bonds, also helps to fix the position of the bound adenine molecule. An ordered water was found in proximity to the bound phosphate ion present in the active site, held by hydrogen bonding to N3 of adenine and Odelta1 of Asp-132. On the basis of the crystal structure, we propose that this water molecule is the nucleophile that participates in the dephosphorylation of the phosphoenzyme intermediate. Solvent isotope effect studies show that there is no rate-determining transfer of a solvent-derived proton in the transition state for the dephosphorylation of the phosphoenzyme intermediate. Such an absence of general base catalysis of water attack is consistent with the stability of the leaving group, namely, the thiolate anion of Cys-13. Consequently, adenine activates the enzyme by binding and orienting a water nucleophile in proximity to the phosphoryl group of the phosphoenzyme intermediate, thus increasing the rate of the dephosphorylation step, a step that is normally the rate-limiting step of this enzymatic reaction.

The bovine protein tyrosine phosphatase (BPTP) is a member of the class of low-molecular weight protein tyrosine phosphatases (PTPases) found to be ubiquitous in mammalian cells. The catalytic site of BPTP contains a CX(5)R(S/T) phosphate-binding motif or P-loop (residues 12-19) which is the signature sequence for all PTPases. Ser19, the final residue of the P-loop motif, interacts with the catalytic Cys12 and participates in stabilizing the conformation of the active site through interactions with Asn15, also in the P-loop. Mutations at Ser19 result in an enzyme with altered kinetic properties with changes in the pK(a) of the neighboring His72. The X-ray structure of the S19A mutant enzyme shows that the general conformation of the P-loop is preserved. However, changes in the loop containing His72 result in a displacement of the His72 side chain that may explain the shift in the pK(a). In addition, it was found that in the crystal, the protein forms a dimer in which Tyr131 and Tyr132 from one monomer insert into the active site of the other monomer, suggesting a dual-tyrosine motif on target sites for this enzyme. Since the activity of this PTPase is reportedly regulated by phosphorylation at Tyr131 and Tyr132, the structure of this dimer may provide a model of a self-regulation mechanism for the low-molecular weight PTPases.

Pyridoxal 5'-phosphate (PLP) binds tightly to bovine low Mr protein tyrosine phosphatase (BPTP), but it is a very poor substrate for the enzyme. The structural basis of this tight binding of PLP is examined here by a variety of methods. Binding constants of a number of PLP analogues were measured with wild-type BPTP, and PLP binding constants of some site-specific mutants of BPTP were determined at pH 5.0 through the use of several independent methods. The tight binding of PLP (Ki = 7.6 microM) causes a downfield shift of the His-72 Cepsilon1H resonance in the 1H NMR spectrum of the protein, consistent with a structural alteration in the phosphate binding loop transmitted through a complex hydrogen bond network that exists between His-72 and Asn-15, which is a residue in the phosphate binding loop. 1H NMR spectroscopy with an MLEV-17 spectral editing scheme was used to monitor the aldehyde resonance of PLP during titration of a catalytically inactive C12A mutant of BPTP. The aldehydic proton resonance of PLP shifted from 10.43 to 10.26 ppm upon complex formation with the C12A mutant. This resonance occurs far from the region where a hemithioacetal hydrogen would be expected to appear, consistent with the conclusion that the Cys-17 side chain of BPTP does not add to the aldehyde group of PLP. UV-visible spectrophotometric titration also supported this conclusion. The binding constant of PLP to a C17A mutant was similar to that exhibited with wild-type protein. These results show that Cys-17 makes virtually no contribution to the tight binding of PLP by BPTP, in contrast to a published report that it is "essential" for binding PLP. On the other hand, Asp-129 of BPTP was found to be very important for binding PLP. It is concluded that Asp-129 binds to the pyridinium nitrogen of PLP and that this renders Asp-129 effectively unavailable to serve its essential catalytic role as a general acid. The interactions described here should be useful in the design of specific inhibitors of this and related phosphotyrosyl protein phosphatases.

In bovine low Mr protein tyrosine phosphatase, the pKa values of His-66 and His-72 are 8.3 and 9.2, respectively. These unusually high values were hypothesized to be caused by electrostatic interactions with several nearby negatively charged groups. To test this, mutant enzymes were made in which one or more carboxylate side chains were removed or introduced near the histidines. Michaelis kinetic parameters, measured using p-nitrophenyl phosphate as a substrate, indicated that all mutant enzymes retained approximately 50% or more of the activity of wild-type enzyme. The effect that each mutation had on the pKa of the nearby histidine was monitored by 1H NMR spectroscopy using the MLEV-17 pulse sequence to filter out the broad interfering amide resonances in the spectra. Independently, computer simulations of the pKas were obtained using the finite difference method to solve the linear Poisson-Boltzmann equation. The proximity of a charged residue to the titrating histidine imidazole largely determined the extent of the pKa perturbation. The change in pKa for His-72 in the mutant enzymes was -1.69 units for D42A, -2.36 units for E23A, -2.99 units for E23A/D42A, and unchanged for E139A and Q143E. Thus, the pKa of His-72 in the double mutant E23A/D42A decreased to nearly that of a free histidine imidazole group. The His-66 pKa change was -1.25 units for E139A and was not significant for the other mutants. His-66, Glu-139, and Gln-143 are at the protein surface and much more exposed to the higher solvent dielectric compared to His-72, Glu-23, and Asp-42. These structural characteristics explain the smaller decrease in the observed pKa of His-66 for the E139A mutant compared to the decrease in the pKa of His-72 when a single nearby carboxylate was removed. These observations were adequately predicted by theoretical electrostatic calculations using the Poisson-Boltzmann equation as a model for a solute molecule of low dielectric in solution of high dielectric.

The early transition metal oxoanions vanadate, molybdate, and tungstate are widely used inhibitors for phosphatase enzymes. These oxoanions could inhibit such enzymes by simply mimicking the tetrahedral geometry of phosphate ion. However, in some cases, the enzyme-inhibitor dissociation constants (Ki) for these oxoanions are much lower than that for phosphate. Such observations gave rise to the hypothesis that in some cases these transition metal oxoanions may inhibit phosphomonoesterases by forming complexes that resemble the trigonal bipyramidal geometry of the SN2(P) transition state. As a test of this, the crystal structures of a low molecular weight protein tyrosine phosphatase at pH 7.5 complexed with the inhibitors vanadate and molybdate were solved at 2.2 A resolution and compared to a newly refined 1.9 A structure of the enzyme. Geometric restraints on the oxoanions were relaxed during refinement in order to minimize model bias. Both inhibitors were bound at the active site, and the overall protein structures were left unchanged, although some small but significant side chain movements at the active site were observed. Vanadate ion formed a covalent linkage with the nucleophile Cys12 at the active site and exhibited a trigonal bipyramidal geometry. In contrast, simple tetrahedral geometry was observed for the weaker molybdate complex. These studies are consistent with the conclusion that vanadate inhibits tyrosine phosphatases by acting as a transition state analog. The structure of the vanadate complex may be expected to closely resemble the transition state for reactions catalyzed by protein tyrosine phosphatases.

The structure of the specific phosphate binding loop (P-loop) of bovine protein tyrosine phosphatase (BPTP) is very similar to that present in high M(r) PTPases. Site-directed mutagenesis was used to explore the role of several conserved residues involved in forming the P-loop of BPTP. Thus, Ser-19 and Ser-43 were individually mutated to alanines, and Asn-15 was mutated to alanine and glutamine. The 1H NMR spectra of the mutants showed good conservation of global secondary structure when compared to wild-type enzyme. Kinetic measurements revealed that only S19A and N15A had substantially altered catalytic activities toward p-nitrophenyl phosphate at pH 5.0, with both mutants exhibiting Vmax values that were 0.25-0.33% of wild-type enzyme. Further kinetic analyses of the N15A and S19A mutants were performed using phosphomonoester substrates with varied phenolic leaving groups. For S19A, the slope of the correlation between Vmax and the substrate leaving group pKa was significantly altered, consistent with a change of the rate-determining step from dephosphorylation to phosphorylation. This was confirmed by partitioning experiments employing methanol as an alternative nucleophile in the dephosphorylation step. Thus, mutating Ser-19 to alanine reduced the efficiency of nucleophilic attack by Cys-12. It is concluded that Ser-19 acts to facilitate the ionization and orientation of Cys-12 for optimal reaction as a nucleophile and as a leaving group. It also appears that Asn-15, Ser-19, His-72, and to a lesser extent Ser-43 serve structural functions that allow the active site to adopt an optimal geometry for phosphate binding. The Asn-15 to Ala mutation appears to disrupt the hydrogen-bonding network, with an accompanying alteration of the geometry of the P-loop. These conclusions are also consistent with changes in the stability of the respective proteins, as measured by urea denaturation.

Two distinct isoenzymes of the human red cell-type acid phosphatase (RCAP) have been known to exist for some time, but the genetic basis of this phenomenon was uncertain. We previously reported the isolation and characterization of two cDNA clones for human RCAP. We showed that the coding regions of the two cDNAs for the human isoenzymes were identical except for a divergent segment spanning nucleotides 176-274, called the variable region. We have now cloned, characterized, and mapped the gene encoding the red cell-type acid phosphatase isoenzymes. The human ACP1 gene is shown to span about 18 kb and to consist of seven exons. The promoter region of ACP1 is very GC-rich and has no apparent TATA or CCAAT boxes. The sequence information confirms that the variable regions of the isoenzymes exist in the gDNA sequence as separate and distinct exons, apparently subject to mutually exclusive alternative splicing. Using a genomic ACP1 clone, we have established the chromosomal localization of the gene to the distal portion of 2p25 by fluorescence in situ hybridization.

Protein tyrosine phosphatases (PTPs) are important enzymes involved in signal transduction, cell cycle regulation, and the control of differentiation. Despite the importance of this class of enzymes in the control of critical cell processes, very little structural information is available for this family of proteins. In this paper, we present the first solution structure of a protein tyrosine phosphatase. This protein is a low molecular weight cytosolic PTP that was initially isolated from bovine heart. The structure that was determined from 1747 NMR-derived restraints consists of a central four-stranded parallel beta-sheet surrounded by four alpha-helices and a short 3(10) helix. The phosphate binding site, identified by chemical shift changes upon the addition of the competitive inhibitors phosphate and vanadate, is in a loop region connecting the C-terminal end of the first beta-strand with the first alpha-helix. Residues in the second, fourth, and fifth alpha-helices and in some of the loop regions connecting the elements of regular secondary structure also contribute to the binding site. The structure determined here is consistent with previous mutagenesis and chemical modification studies conducted on this protein.

The first X-ray crystallographic structure of a member of the class of low molecular weight (M(r) 18,000) phosphotyrosyl phosphatases is presented. Bovine heart phosphotyrosyl phosphatase (BHPTP) exemplifies this class and is highly homologous (94% sequence identity) to an isoenzyme known as red cell acid phosphatase that is present throughout human tissues. The high-resolution (2.2-A) crystal structure of BHPTP shows that the enzyme consists of a four-strand central parallel beta sheet with alpha helices packed on both sides in a manner characteristic of a Rossmann fold. A bound phosphate ion defines the active site location in a loop of the first beta alpha beta motif at the C-terminus of the beta sheet. The location and enzymatic significance of the residues in the characteristic low molecular weight PTPase active site motif, including the essential arginine (Arg 18) and nucleophilic cysteine (Cys 12), are described. The functional role of a histidine (His 72) suggested previously to be near the active site is defined in the structure, as well as a potential proton donor for the leaving group in the tyrosyl phosphate cleavage. Surface maps of BHPTP define a hydrophobic crevice suitable for phosphotyrosyl peptide binding. Comparison of the BHPTP structure to the related, but structurally distinct enzyme PTP1B is made, illustrating the unique way this smallest of these phosphatases has formed the phosphotyrosine active site.

Phosphotyrosyl protein phosphatases play an important role in mediating cellular signal transduction; yet three-dimensional structures of this important class of proteins have not been reported. We present the sequence-specific 1H, 13C, and 15N backbone assignments for the low molecular weight bovine heart phosphotyrosyl protein phosphatase (BHPTPase) (157 residues, 17,900). The assignments were obtained from a combination of double- and triple-resonance multidimensional NMR experiments. From these assignments, the secondary structure of BHPTPase was determined from an analysis of NOE patterns, 3JHNH alpha coupling constants, 13C alpha and 13CO chemical shifts, and amide 1H exchange rates. BHPTPase was found to consist of a four-stranded parallel beta-sheet (residues K6-C12, W39-A45, Y87-M91, and K112-L116), four alpha-helices (residues I21-D32, R58-G67, S94-N104, and D135-R157), and one stretch of beta 10-helix (residues K79-F85). The secondary structure is characteristic of the beta alpha beta structural motif. The secondary structure elements identified in this study are consistent with previous chemical and mutagenesis studies of BHPTPase structure.

Two crystal forms of bovine heart phosphotyrosyl protein phosphatase (BHPTP) have been examined by X-ray analysis. One crystal form grows as long rods with triclinic crystal symmetry and diffracts to 3 A resolution. The diffraction pattern of this form of the crystal shows twinning about a major axis. A second crystal form of BHPTP grows as flat trapezoidal prisms with monoclinic symmetry C2, and unit cell parameters a = 95.3 A, b = 43.3 A, c = 41.2 A and beta = 113.5 degrees. The unit cell dimensions indicate that there is one 18 kDa molecule per asymmetric unit. These crystals diffract to at least 2.2 A resolution and are resistant to decay in the X-ray beam.

Earlier covalent modification experiments indicated that histidine, arginine, and carboxylic acid residues were involved in the catalytic mechanism of the acid phosphatase enzyme from human prostate. The present study utilizes site-directed mutagenesis to evaluate the catalytic importance of 7 residues of human prostatic phosphatase, namely His12, His257, Asp258, Arg11, Arg15, Arg54, and Arg79, which are highly conserved in the sequences of high molecular weight acid phosphatases. Both wild type and mutant enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions to a leader peptide of prepro-alpha-factor, which resulted in the secretion of processed proteins into the culture media. The kinetic parameters of the recombinant wild type enzyme were shown to be effectively identical with those of the native protein. The circular dichroism spectra of wild type and mutant proteins were effectively superimposable. Replacement of His12, which was previously shown to be transiently phosphorylated during catalysis, as well as the adjacent Arg11, both resulted in > 10(4) reductions of enzyme activity. Consistent with a postulated involvement of arginine residues in substrate binding, large reductions in Vmax caused by the R15A and R79A mutations were accompanied by marked increases in Km. However, the mutation R54A had only small effects on kinetic parameters. Essential roles for both His257 and Asp258 were also established. The D258A mutation not only caused larger decreases of Vmax toward p-nitrophenyl phosphate compared to the effect of the H257A mutation, but it also caused the reaction to become more sensitive to the nature of the leaving group. It is concluded that His12 and Asp258 are involved in the formation of the phosphoenzyme intermediate as an acceptor of the phospho group and a proton donor for the substrate leaving group, respectively, while His257 may participate in substrate binding or may facilitate the breakdown of the phosphoenzyme by maintaining the protonated state of the phosphorylated His12 residue.

The roles of the 8 conserved cysteines and 1 arginine in the low molecular weight phosphotyrosyl protein phosphatases were investigated using site-directed mutagenesis of the recombinant bovine heart enzyme. Single mutants of cysteine to serine were studied for each cysteine; alanine replacements were also made for Cys-12, Cys-17, and Arg-18. The CD spectra of the purified proteins were effectively superimposable, consistent with the conclusion that no major structural alterations had occurred, but 1H NMR spectroscopy did reveal some spectral shifts in the aromatic region. Kinetic analysis of the mutant proteins demonstrated that only Cys-12, Cys-17, and Arg-18 had significantly altered catalytic activity toward the substrate p-nitrophenyl phosphate at pH 5. The Cys-12 and Arg-18 mutants were effectively inactive. Thus, it is concluded that Cys-12 is the catalytic nucleophile, and Arg-18 presumably serves an essential function in substrate binding. The C17S mutant had 6% residual activity compared with wild type protein, whereas the C17A mutant had 37% activity. Consistent with the observed activity of the Cys-17 mutant, a covalent phosphocysteine intermediate was trapped and identified by 31P NMR. Further kinetic analysis of C17A using several aryl phosphate monoester substrates with different leaving group pK alpha values indicated that no change in the rate-determining step of the catalytic mechanism had occurred, that is, dephosphorylation of the covalent phosphoenzyme intermediate remains rate-limiting. The C17A mutant had a 4-fold higher phosphate Ki and slightly higher Km values for p-nitrophenyl phosphate suggesting that Cys-17 may be important for optimal positioning of the substrate phosphate moiety.

Product inhibition and 18O exchange experiments suggest that the Yersinia protein-tyrosine phosphatase-catalyzed phosphate monoester hydrolysis proceeds through at least two different chemical steps, i.e. the formation and breakdown of a covalent phosphoenzyme intermediate. The pH dependence of kcat values is bell-shaped, with the apparent pKa derived from the acidic limb of the profile at 4.6 for both p-nitrophenyl phosphate and beta-naphthyl phosphate, whereas the apparent pKa derived from the basic limb of the profile is substrate-dependent, with apparent pKa values of 5.2 and 5.8 for p-nitrophenyl phosphate and beta-naphthyl phosphate, respectively. Twelve aryl phosphates with leaving groups having pKa values from approximately 7 to 10 are also examined as substrates at two pH values. At pH 4.0, the beta lg values is effectively zero, whereas at pH 7.5, a beta lg value of 0.16 is observed. Collectively, our results suggest that the rate-determining step under acidic conditions corresponds to the breakdown of the phosphoenzyme intermediate, whereas under more alkaline conditions, substrate effects also contribute to the rate-limiting step. A model is proposed for the mechanism of the Yersinia protein-tyrosine phosphatase-catalyzed reaction.

The role of the two conserved histidine residues in the low-molecular-weight phosphotyrosyl protein phosphatases was investigated using site-directed mutagenesis of the recombinant bovine heart enzyme. His-66 and His-72 were individually mutated to alanine and to asparagine. A double mutant, containing only alanines in place of the histidines, was also created. The 1H NMR spectra of the purified proteins revealed no apparent tertiary structure alterations. Microscopic pKas for the two histidines were determined from a pH titration of the wild-type enzyme using 1H NMR spectroscopy and an MLEV-17 spectral editing scheme to more readily follow shifts in the specific histidine resonance peaks. His-66 titrates with an apparent pKa of 8.4 while for His-72 the value is 9.2. Since earlier chemical modification experiments indicated that the wild-type enzyme was inactivated by the histidine-selective modification reagent diethyl pyrocarbonate (DEP), the histidine mutants were tested for sensitivity to DEP. Both of the histidine single mutants were inactivated by DEP, and surprisingly, the double mutant containing no histidines was also readily inactivated by DEP. Thus, for this protein, modification by DEP is not specific for histidine residues. Kinetic studies of the mutant proteins reveal that neither histidine is essential in the catalytic mechanism. His-66 mutants showed virtually identical catalytic properties compared to wild-type enzyme, whereas His-72 mutants had reduced specific activity and higher phosphate Ki and lower Km values at pH5 and higher. It is proposed that His-72, although not essential for catalysis, may serve a significant structural role at the enzyme active site.

Site-directed mutagenesis was used to explore the role of potential proton donors in the mechanism of the Escherichia coli acid phosphatase that is encoded by the appA gene. Asp304 appeared to be the only carboxylic acid residue that is conserved in the protein sequences of the high molecular weight acid phosphatases. The mutations Asp304Ala and Asp304Glu were introduced into appA and the corresponding proteins were overexpressed in E. coli and purified to homogeneity. Only small decreases were observed for the Km values of the substrates p-nitrophenyl phosphate, fructose 1,6-diphosphate, and tripolyphosphate. However, Vmax was greatly decreased, and the magnitude of effect depended markedly on substrate. Both mutant proteins exhibited significantly lower Vmax values with fructose 1,6-diphosphate, which possesses a much poorer leaving group than do the other two substrates. The importance of the leaving group was further tested by using a number of phenyl and alkyl phosphate derivatives as substrates. A linear correlation was observed between log Vmax and the pKa of the substrate leaving group for catalysis by the Asp304Ala mutant enzyme. These results are consistent with partition experiments using ethylene glycol as an alternate nucleophile, which indicated that for the Asp304Ala protein, the formation of a phosphoenzyme intermediate is the rate-determining step, in contrast to the situation for the wild type enzyme and the His303Ala mutant. In the latter case, the rate-limiting step of the reaction is interpreted to be the breakdown of phosphoenzyme. It is concluded that Asp304, rather than His303, is involved in protonation of the substrate leaving group.