Substance P as a transglutaminase substrate: identification of the reaction products by fast atom bombardment mass spectrometry

Substance P was found to be an effective acyl donor substrate of transglutaminase in vitro, the reaction products having been examined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fast atom bombardment mass spectrometry. Electrophoretic experiments showed that Substance P incorporated 14C-labeled polyamines when incubated with purified guinea pig liver transglutaminase and Ca2+. Extensive use of fast atom bombardment mass spectrometry allowed to establish that: i) a 1:1 adduct Substance P-spermine is formed; ii) only a single glutamine residue out of two, i.e. Gln-5, acts as acyl donor, iii) the single lysine residue of the neuropeptide is unable to act as acyl acceptor. A direct analytical methodology to detect transglutaminase reaction products is described.     

Enzymatic synthesis of vasoactive intestinal peptide analogs by transglutaminase.

Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln16, which was identified as the amino acceptor substrate. Different gamma(glutamyl16)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono-dansylcadaverine of the peptide were purified by high-performance liquid chromatography on a reverse-phase column and were analyzed by electrospray mass spectrometry. When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys21, was demonstrated to be involved in both inter- and intramolecular cross-link formation.     

A Pro --> Ala substitution in melittin affects self-association, membrane binding and pore-formation kinetics due to changes in structural and electrostatic properties

Melittin, the main component of bee venom of Apis mellifera, contains a proline at position 14, which is highly conserved in related peptides of various bee venoms. To investigate the structural and functional role of Pro14 a melittin analogue was studied where proline is substituted by an alanine residue (P14A). The investigations were focussed on: (i) the secondary structure in aqueous solution and membranes; (ii) the self-association in solution; (iii) the binding to POPC membranes; and (iv) the P14A-induced leakage and pore formation in membrane vesicles. Circular dichroism and gel filtration experiments showed that P14A exists at concentrations < 12 microM in monomeric form with an alpha-helicity of 28 +/- 7%. A further increase in peptide concentration leads to the formation of large aggregates consisting of 9 +/- 1 monomers. While binding studies with POPC vesicles revealed for P14A a stronger binding affinity towards membranes than for melittin, the peptide-induced leakage of fluorescent markers from vesicles was less efficient for P14A than for melittin. Furthermore, an unexpected efflux behaviour at high values of bound P14A was observed which indicated that the pore formation kinetics for P14A is more complex than it was reported for melittin. The different features of P14A in aggregation, binding and efflux compared to melittin are mainly ascribable directly to structural changes caused by the proline --> alanine substitution. Furthermore, the results indicate an improved screening of the positively charged residues of P14A by counterions which contributes additionally to the observed differences in peptide activities. It is suggested that the presence of proline in melittin is not only of structural importance but also influences indirectly the electrostatic properties of the native peptide.     

Structural features of glutamine substrates for human plasma factor XIIIa (activated blood coagulation factor XIII)

The action of human plasma factor XIIIa (thrombin-activated blood coagulation factor XIII) and guinea pig liver transglutaminase on purified caseins, fibrin, the derivatized gamma chain of fibrin, and a number of synthetic glutamine peptides, and peptide derivatives is reported. There are wide variations in the properties of the individual proteins and peptides as substrates for amine incorporation by the two transglutaminases. beta-Casein and several of its derivatives are excellent substrates for factor XIIIa. However, beta-casein is a relatively poor substrate for the liver enzyme. The primary site of amine incorporation by factor XIIIa in beta-casein was identified as glutamine 167. This was accomplished by labeling with fluorescent amine followed by proteolytic digestion and identification of labeled peptides. An 11-residue peptide and a 15-residue peptide, each containing 1 glutamine residue and each modeled after the primary site of amine incorporation in beta-casein, were prepared. A 13-residue peptide modeled after the primary crosslinking site in fibrin gamma chain was also prepared. Each of these polypeptides proved to be an efficient substrate for factor XIIIa and displayed significantly better substrate properties than a number of small glutamine peptide derivatives that are good substrates for liver transglutaminase.     

Cross-linking of alpha 2-plasmin inhibitor to fibrin catalyzed by activated fibrin-stabilizing factor

During blood coagulation alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked with fibrin by an activated fibrin-stabilizing factor (FSFa) plasma transglutaminase, activated coagulation factor XIII). When alpha 2PI was treated with FSFa in the absence of acceptor amino groups, the inhibitor lost more than 90% of its capacity to be cross-linked to fibrin because of hydrolysis of the gamma-carboxamides of FSFa-susceptible glutamine residues. Chemical modifications of the inhibitor's lysine epsilon-amino groups did not affect the cross-linking capacity of the inhibitor with fibrin, whereas the same chemical modifications in fibrinogen resulted in a remarkable loss of cross-linking capacity. These observations suggest that alpha 2PI plays a role as an acyl donor with its FSFa-susceptible glutamine residues in the cross-linking reaction with fibrin, and fibrin serves as an acyl acceptor with its lysine residues. The number of FSFa-susceptible glutamine residues/molecule of the inhibitor was estimated by measuring the maximum incorporation of [3H]histamine into the inhibitor and by analyzing the distribution of radioactivity in a tryptic digest of [14C]histamine-incorporated alpha 2PI.l It was found that each inhibitor molecule has one glutamine residue that is most susceptible to FSFa. When the radioactive histamine-incorporated inhibitor was reacted with excess amounts of plasmin, a small fragment carrying all the released radioactivity was rapidly released from the NH2-terminal part of the inhibitor moiety of the complex. The NH2-terminal amino acid sequence of the inhibitor was analyzed before and after treatment with FSFa or before and after incorporation of radioactive histamine. The glutamine residue at the second position from the NH2-terminal end was converted to a glutamic acid residue when the inhibitor was treated with FSFa. When the radioactive histamine-incorporated inhibitor ws analyzed, the radioactivity was found predominantly at the second position from the NH2-terminal end. These results indicate that the glutamine residue susceptible to FSFa in alpha 2PI is located next to the NH2-terminal residue.     

Transglutaminase-catalyzed cross-linking through diamines and polyamines.

Transglutaminases were found to catalyze the formation of cross-links between peptide chains by means of a transfer reaction between the carboxamide group of a glutamine residue in each chain and both primary amino groups of a diamine or a polyamine. Production of this heretofore undescribed linkage by guinea pig liver transglutaminase was demonstrated by the use of high performance liquid chromatography in a model system using glutamine peptide derivatives and a variety of diamines and polyamines. Evidence for intermolecular cross-linking through polyamines with both the liver enzyme and thrombin-activated human plasma blood coagulation factor XIII was obtained by the use of a guanidinated derivative of beta-casein.     

Structural features of glutamine substrates for transglutaminases. Role of extended interactions in the specificity of human plasma factor XIIIa and of the guinea pig liver enzyme

Amino acid residues at several locations in close primary vicinity to a substrate glutamine residue have been recognized as important determinants for the specificities of human plasma factor XIIIa and guinea pig liver transglutaminase (Gorman, J. J., and Folk, J. E. (1981) J. Biol. Chem. 256, 2712-2715). The present studies measure the influence on transglutaminase specificity of some changes in amino acid side chains in a small synthetic glutamine peptide amide, Leu-Gly-Leu-Gly-Gln-Gly-Lys-Val-Leu-GlyNH2, which was designed to contain most of the known elements needed for enzyme recognition. The results are in agreement with previous findings and show that full catalytic activity of each enzyme may be retained upon replacement of the lysine residue by certain other amino acid residues. Evidence is provided that serine in place of glycine at one or more positions causes a significant increase in specificity with factor XIIIa, but not with liver enzyme. The effective substrate property for factor XIIIa seen with the model peptide amide is lost upon reversal of the sequence Val-Leu. This is not the case with the liver enzyme even though replacement of either of these amino acids by alanine causes a pronounced loss in activity with this enzyme. These differences and the effects of various other substitutions in the model peptide amide on the enzymes' specificities points up the relatively stringent structural requirements of factor XIIIa and the rather broad requirements for liver transglutaminase.     

The carboxy-terminal lysine of alpha B-crystallin is an amine-donor substrate for tissue transglutaminase.

A hexapeptide, corresponding to the sequence around the glutamine in beta A3-crystallin that functions as amine-acceptor for transglutaminase, was synthesized. This peptide was biotinylated and used as a probe to identify amine-donor substrates for transglutaminase among lens proteins. It was found that Ca(2+)-activated transglutaminase linked this peptide not only to several beta-crystallins but, unexpectedly, also to alpha B-crystallin. The C-terminal lysine residue of alpha B-crystalline could be identified as the site of linkage. This strengthens the notion that, at least in crystallins, all transglutaminase substrate residues are located in terminal extensions of the polypeptides. It was shown that in lens homogenate, alpha B-crystallin can be covalently crosslinked to beta-crystallins by transglutaminase. The transglutaminase-mediated crosslinking of alpha B-crystallin may have implications for its involvement in normal and pathological processes in lens and other tissues.     

Influence of the lipid composition on the kinetics of concerted insertion and folding of melittin in bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the ¹⁹Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the ¹⁹Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an α-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of ¹⁹Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the α-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.     

Transglutaminase-catalyzed modifications of SV-IV, a major protein secreted from the rat seminal vesicle epithelium

One of the major proteins secreted from the rat seminal vesicle epithelium, namely SV-IV, was shown to act in vitro as acyl donor and acceptor substrate for transglutaminase from both guinea pig liver and rat anterior prostate secretory fluid. Electrophoretic and chromatographic experiments indicated that the enzyme catalyzed the formation of multiple modified forms of SV-IV. In the absence of small Mr amines, transglutaminase was able to produce at least six different molecular forms of the protein, half of which possessed an Mr higher than that of native SV-IV. These findings suggested that a variable number of intermolecular, and perhaps intramolecular, crosslinks were formed between one or both glutamine residues and one or more lysine residues occurring in the SV-IV polypeptide chain. In addition, at least three modified forms of the protein were produced by transglutaminase in the presence of high concentrations of spermidine, thus indicating the formation of different (gamma-glutamyl)polyamine derivatives of SV-IV. Rabbit uteroglobin and rat anterior prostate secretory protein(s) were also shown to be able to covalently bind spermidine in the presence of the enzyme. The possible biological significance of transglutaminase-mediated modifications of SV-IV, as well as of other proteins occurring in the mammal seminal fluid, are discussed.     

N-bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11

Neutral endopeptidase 24.11 is rapidly inactivated by N-bromoacetyl-D-leucylglycine in a reaction which follows first-order kinetics at pH 8 and 37 degrees C. The concentration dependence of inactivation revealed saturation kinetics with an apparent Ki of 10 mM and kappa inact of 0.4 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by either the substrate Leu5-enkephalin or the competitive inhibitors Phe-Gly or Phe-Ala. Inactivation of enzyme by N-bromo-[14C]acetyl-D-leucylglycine proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Tryptic digestion of the radioactively labeled enzyme followed by high performance liquid chromatography allowed the isolation of a modified peptide with the sequence T-D-V-H-S-P-G-N-F-R in which histidine (His704) is the modified residue. Site-directed mutagenesis was used to generate a mutant form of the enzyme in which histidine 704 was converted to a glutamine residue. This mutant enzyme retained less than 0.1% of the activity of the native enzyme. These results demonstrate that His704 is at the active site of neutral endopeptidase 24.11 and suggest a catalytic role for this residue.     

Influence of lipid chain unsaturation on membrane-bound melittin: a fluorescence approach

Melittin, a cationic hemolytic peptide, is intrinsically fluorescent due to the presence of a single functionally important tryptophan residue. The organization of membrane-bound melittin is dependent on the physical state and composition of membranes. In particular, polyunsaturated lipids have been shown to modulate the membrane-disruptive action of melittin. Phospholipids with polyunsaturated acyl chains are known to modulate a number of physical properties of membranes and play an important role in regulating structure and function of membrane proteins. In this study, we have used melittin to address the influence of unsaturated lipids in modulating lipid-protein interactions. Our results show that fluorescence parameters such as intensity, emission maximum, polarization, lifetime and acrylamide quenching of melittin incorporated in membranes are dependent on the degree of unsaturation of lipids in membranes. Importantly, melittin in membranes composed of various unsaturated lipids shows red edge excitation shift (REES) implying that melittin is localized in a motionally restricted region in membranes. The extent of REES was found to increase drastically in membranes with increasing unsaturation, especially when the lipids contained more than two double bonds. In addition, increasing unsaturation in membranes causes a considerable change in the secondary structure of membrane-bound melittin. Taken together, our results assume significance in the overall context of the role of unsaturated lipids in membranes in the organization and function of membrane proteins and membrane-active peptides.     

Proton NMR studies of the biologically active 1-34 fragment of bovine parathyroid hormone: examination of a structural model

Proton NMR spectra of the biologically active 1-34 fragment of bovine parathyroid hormone (bPTH) were studied as a function of pH over the range of pH 4 to 10, in buffer and in 6 M guanidine DC1. One of the histidine C-2 peaks titrated normally, with a pKa value of 6.8, but the other two histidines in this peptide had pKa values of 6.3. Denatured PTH showed only one histidine C-2 peak with a pKa of 6.7. An aliphatic peak identified as due to either a methionine or a glutamine residue also shifted with pH, and the pKa for this shift was 6.3. Finally, small but significant upfield shifts in the methyl and methylene resonances were observed as a function of pH, and when compared to the denatured peptide. These results indicate that the N-terminal domain of native PTH has considerable structure in solution, and are consistent with a theoretical model for the folding of this peptide.     

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.     

Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.

Molecular dynamics trajectories of melittin in an explicit dimyristoyl phosphatidylcholine (DMPC) bilayer are generated to study the details of lipid-protein interactions at the microscopic level. Melittin, a small amphipathic peptide found in bee venom, is known to have a pronounced effect on the lysis of membranes. The peptide is initially set parallel to the membrane-solution interfacial region in an alpha-helical conformation with unprotonated N-terminus. Solid-state nuclear magnetic resonance (NMR) and polarized attenuated total internal reflectance Fourier transform infrared (PATIR-FTIR) properties of melittin are calculated from the trajectory to characterize the orientation of the peptide relative to the bilayer. The residue Lys7 located in the hydrophobic moiety of the helix and residues Lys23, Arg24, Gln25, and Gln26 at the C-terminus hydrophilic form hydrogen bonds with water molecules and with the ester carbonyl groups of the lipids, suggesting their important contribution to the stability of the helix in the bilayer. Lipid acyl chains are closely packed around melittin, contributing to the stable association with the membrane. Calculated density profiles and order parameters of the lipid acyl chains averaged over the molecular dynamics trajectory indicate that melittin has effects on both layers of the membrane. The presence of melittin in the upper layer causes a local thinning of the bilayer that favors the penetration of water through the lower layer. The energetic factors involved in the association of melittin at the membrane surface are characterized using an implicit mean-field model in which the membrane and the surrounding solvent are represented as structureless continuum dielectric material. The results obtained by solving the Poisson-Bolztmann equation numerically are in qualitative agreement with the detailed dynamics. The influence of the protonation state of the N-terminus of melittin is examined. After 600 ps, the N-terminus of melittin is protonated and the trajectory is continued for 400 ps, which leads to an important penetration of water molecules into the bilayer. These observations provide insights into how melittin interacts with membranes and the mechanism by which it enhances their lysis.     

Platelet vinculin: a substrate of activated factor XIII

In addition to plasma, Factor XIII of blood coagulation (FXIII) is also present in the cytosol of platelets, monocytes and macrophages. However, its intracellular function has not yet been revealed. Activated Factor XIII (FXIIIa) is a transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) of highly restricted substrate specificity with only a few known protein substrates. In this report, we showed that FXIIIa can link dansylcadaverine, radiolabelled histamine and putrescine to vinculin. Quantitative determinations revealed that in the vinculin molecule a single glutamine residue can serve as acyl donor for the incorporation of small-molecular-weight amines. Vinculin could not be crosslinked to another vinculin molecule. It could be covalently bound, however, to fibrinogen, which indicates that the acyl donor glutamine residue can be engaged in an epsilon-(gamma-glutamyl)lysyl crosslink formation. Since it has been shown that platelet actin and myosin, two main components of cytoskeleton, are also substrates for FXIIIa, and that vinculin is associated to the cytoskeleton during platelet activation, the involvement of FXIII in the stabilization of cytoskeleton at certain phases of cellular function is a likely possibility.     

Further studies on the site-specific protein modification by microbial transglutaminase

A guinea pig liver transglutaminase (G-TGase)-mediated procedure for the site-specific modification of chimeric proteins was recently reported. Here, an alternative method with advantages over the recent approach is described. This protocol utilizes a microbial transglutaminase (M-TGase) instead of the G-TGase as the catalyst. M-TGase, which has rather broad structural requirements as compared to the G-TGase, tends to catalyze an acyl transfer reaction between the gamma-carboxamide group of a intact protein-bound glutamine residue and various primary amines. To demonstrate the applicability of the M-TGase-catalyzed protein modification in a drug delivery system, we have utilized recombinant human interleukin 2 (rhIL-2) as the target protein and two synthetic alkylamine derivatives of poly(ethyleneglycol) (PEG12; MW 12 kDa) and galactose-terminated triantennary glycosides ((Gal)(3))) as the modifiers. For the M-TGase-catalyzed reaction with PEG12 and (Gal)(3), 1 mol of alkylamine was incorporated per mole of rhIL-2, respectively. Peptide mapping of (Gal)(3)-modified rhIL-2 ((Gal)(3)-rhIL-2) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) suggested that the Gln74 residue in rhIL-2 was site specifically modified with (Gal)(3). The PEG12-rhIL-2 and (Gal)(3)-rhIL-2 conjugates retained full bioactivity relative to the unmodified rhIL-2. In pharmacokinetic studies, PEG12-rhIL-2 was eliminated more slowly from the circulation than rhIL-2, whereas (Gal)(3)-rhIL-2 accumulated in the liver via hepatic asialoglycoprotein receptor binding. The results of this study expand the applicability of the TGase-catalyzed methodology for the preparation of protein conjugates for clinical use.     

Consequences of alteration in leucine zipper sequence of melittin in its neutralization of lipopolysaccharide-induced proinflammatory response in macrophage cells and interaction with lipopolysaccharide

The bee venom antimicrobial peptide, melittin, besides showing versatile activity against microorganisms also neutralizes lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells. However, how the amino acid sequence of melittin contributes in its anti-inflammatory properties is mostly unknown. To determine the importance of the leucine zipper sequence of melittin in its neutralization of LPS-induced inflammatory responses in macrophages and interaction with LPS, anti-inflammatory properties of melittin and its three analogues and their interactions with LPS were studied in detail. Two of these analogues, namely melittin Mut-1 (MM-1) and melittin Mut-2 (MM-2), possess leucine to alanine substitutions in the single and double heptadic leucine residue(s) of melittin, respectively, whereas the third analogue is a scrambled peptide (Mel-SCR) that contains the amino acid composition of melittin with minor rearrangement in its leucine zipper sequence. Although MM-1 partly inhibited the production of proinflammatory cytokines in RAW 264.7 and rat primary macrophage cells in the presence of LPS, MM-2 and Mel-SCR were negligibly active. A progressive decrease in interaction of melittin with LPS, aggregation in LPS, and dissociation of LPS aggregates with alteration in the leucine zipper sequence of melittin was observed. Furthermore, with alteration in the leucine zipper sequence of melittin, these analogues failed to exhibit cellular responses associated with neutralization of LPS-induced inflammatory responses in macrophage cells by melittin. The data indicated a probable important role of the leucine zipper sequence of melittin in neutralizing LPS-induced proinflammatory responses in macrophage cells as well as in its interaction with LPS.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (Ka) of 2.85 x 10(6) M(-1) and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (delta G0), -8.8 kcal mol(-1), obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Kinetic and spectroscopic studies of Tritrichomonas foetus low-molecular weight phosphotyrosyl phosphatase. Hydrogen bond networks and electrostatic effects

Virtually all of the eukaryotic low-molecular weight protein tyrosine phosphatases (LMW PTPases) studied to date contain a conserved, high-pK(a) histidine residue that is hydrogen bonded to a conserved active site asparagine residue of the phosphate binding loop. However, in the putative enzyme encoded by the genome of the trichomonad parasite Tritrichomonas foetus, this otherwise highly conserved histidine is replaced with a glutamine residue. We have cloned the gene, expressed the enzyme, demonstrated its catalytic activity, and examined the structural and functional roles of the glutamine residue using site-directed mutagenesis, kinetic measurements, and NMR spectroscopy. Titration studies of the two native histidine residues in the T. foetus enzyme as monitored by (1)H NMR revealed that H44 has a pK(a) of 6.4 and H143 has a pK(a) of 5.3. When a histidine residue was introduced in place of the native glutamine at position 67, a pK(a) of 8.2 was measured for this residue. Steady state kinetic methods were employed to study how mutation of the native glutamine to alanine, asparagine, and histidine affected the catalytic activity of the enzyme. Examination of k(cat)/K(m) showed that Q67H exhibits a substrate selectivity comparable to that of the wild-type (WT) enzyme, while Q67N and Q67A show reduced activity. The effect of pH on the reaction rate was examined. Importantly, the pH-rate profile of the WT TPTP enzyme revealed a much more clearly defined acidic limb than that which can be observed for other wild-type LMW PTPases. The pH-rate curve of the Q67H mutant shows a shift to a lower pH optimum relative to that seen for the wild-type enzyme. The Q67N and Q67A mutants showed curves that were shifted to higher pH optima. Although the active site of this enzyme is likely to be similar to that of other LMW PTPases, the hydrogen bonding and electrostatic changes afford new insight into factors affecting the pH dependence and catalysis by this family of enzymes.