Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of k_cat^app/K_M probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the K_M and k_cat^app are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of k_cat^app, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.     

A lysine conserved in the monoamine oxidase family is involved in oxidation of the reduced flavin in mouse polyamine oxidase

Lysine 315 of mouse polyamine amine oxidase corresponds to a lysine residue that is conserved in the flavoprotein amine oxidases of the monoamine oxidase structural family. In several structures, this lysine residue forms a hydrogen bond to a water molecule that is hydrogen-bonded to the flavin N(5). Mutation of Lys315 in polyamine oxidase to methionine was previously shown to have no effect on the kinetics of the reductive half-reaction of the enzyme (M. Henderson Pozzi, V. Gawandi, P.F. Fitzpatrick, Biochemistry 48 (2009) 1508-1516). In contrast, the mutation does affect steps in the oxidative half-reaction. The k_cat value is unaffected by the mutation; this kinetic parameter likely reflects product release. At pH 10, the k_cat/K_m value for oxygen is 25-fold lower in the mutant enzyme. The k_cat/K_O2 value is pH-dependent for the wild-type enzyme, decreasing below a pK_a of 7.0, while this kinetic parameter for the mutant enzyme is pH-independent. This is consistent with the neutral form of Lys315 being required for more rapid flavin oxidation. The solvent isotope effect on the k_cat/K_O2 value increases from 1.4 in the wild-type enzyme to 1.9 in the mutant protein, and the solvent inventory changes from linear to bowed. The effects of the mutation can be explained by the lysine orienting the bridging water so that it can accept the proton from the flavin N(5) during flavin oxidation. In the mutant enzyme the lysine amine would be replaced by a water chain.     

Investigation of cobalt(II) substituted carboxypeptidase a interacting with azide and cyanate ions

Cobalt(II)-substituted carboxypeptidase A has been found to reversibly bind N3^? and NCO^?, but not NCS^?, in the pH range 5–10, thus including the pH range of activity of the enzyme. The pH dependence of the anion binding constant is affected by two ionizations, which are assigned as those regulating k_cat and K_M. The electronic and ₁H NMR spectra are consistent with a substantially pseudotetrahedral geometry of the anion derivatives.     

Dependence on pH of the activity of pig liver esterase

The dependence on pH of the kinetic parameters for the hydrolysis of phenyl acetate catalyzed by pig liver carboxylesterase was examined for purified high-isoelectric point and low-isoelectric point fractions of enzyme that were separated by isoelectric focusing. The values of k_cat are half-maximal at pH 4.3 and 5.1 for the high- and low-isoelectric point forms, respectively, and show a shallow dependence on pH with a value of n = 0.5. The absence of a change in the pH dependence of k_cat for the high-isoelectric point enzyme in the presence of high concentrations of methanol, which reacts with the acetyl-enzyme intermediate to give methyl acetate, provides evidence that the pH dependence is not caused by a change in rate-determining step. This means that if an imidazole group is involved in catalysis its pK must be perturbed downward by 2–3 units. The pH dependence of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ is biphasic with apparent pK values for dissociations of the free enzyme near 7 and 4 for both the high- and low-isoelectric point enzymes. Inhibition by a second molecule of substrate and by methanol are strongest for high-pH forms of the enzyme.     

Cloning,expression and some properties of α-carbonic anhydrase from Helicobacter pylori

The α-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO₂ hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are k_cat=2.4×10⁵ s⁻¹, K_M=17 mM and k_cat/K_M=1.4×10⁷ M⁻¹ s⁻¹. The pH dependence of k_cat/K_M fits with a simple titration curve with pK_a=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO₂ hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, k_enz, of 24 M⁻¹ s⁻¹ at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a k_enz value of 665 M⁻¹ s⁻¹ was obtained under similar conditions.     

Catalytic role of the calcium ion in GH97 inverting glycoside hydrolase

The role of calcium ion in the active site of the inverting glycoside hydrolase family 97 enzyme, BtGH97a, was investigated through structural and kinetic studies. The calcium ion was likely directly involved in the catalytic reaction. The pH dependence of k_cat/K_m values in the presence or absence of calcium ion indicated that the calcium ion lowered the pK_a of the base catalyst. The significant decreases in k_cat/K_m for hydrolysis of substrates with basic leaving groups in the absence of calcium ion confirmed that the calcium ion facilitated the leaving group departure.     

Solvent isotope and viscosity effects on the steady-state kinetics of the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of a broad range of primary and secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. With nitroethane as substrate the ^D2O(k_cat/K_M) value is 0.6 and the ^D2Ok_cat value is 2.4. The k_cat proton inventory is consistent with a single exchangeable proton in flight, while the k_cat/K_M is consistent with either a single proton in flight in the transition state or a medium effect. Increasing the solvent viscosity did not affect the k_cat or k_cat/K_M value significantly, establishing that nitroethane binding is at equilibrium and that product release does not limit k_cat.     

Steady-state kinetic mechanism of LodA,a novel cysteine tryptophylquinone-dependent oxidase

LodA is a novel lysine-ε-oxidase which possesses a cysteine tryptophylquinone cofactor. It is the first tryptophylquinone enzyme known to function as an oxidase. A steady-state kinetic analysis shows that LodA obeys a ping-pong kinetic mechanism with values of k_cat of 0.22 ± 0.04 s⁻¹, K_lysine of 3.2 ± 0.5 μM and K_O2 of 37.2 ± 6.1 μM. The k_cat exhibited a pH optimum at 7.5 while k_cat/K_lysine peaked at 7.0 and remained constant to pH 8.5. Alternative electron acceptors could not effectively substitute for O₂ in the reaction. A mechanism for the reductive half reaction of LodA is proposed that is consistent with the ping-pong kinetics.     

Chemical modification of xylanase from alkalothermophilic Bacillus species: evidence for essential carboxyl group

The role of carboxyl group in the catalytic action of xylanase (M_r 35 000) from an alkalothermophilic Bacillus sp. was delineated through iinetic and chemical modification studies using Woodward's Reagent K. The kinetics of inactivation indicated that one carboxyl residue was essential for the xylanase activity with a second order rate constant of 3300 M⁻¹ min⁻¹. The spectrophotometric analysis at 340 nm revealed that the inhibition was correlated with modification of 24 carboxyl residues. In the presence of protecting ligand, modification of one carboxyl group was prevented. The pH profile showed apparent pK values of 5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for enzyme-substrate complex. The pH dependence of inactivation was consistent with the modification of carboxyl group. The kinetic analysis of the modified enzyme showed similar K_m and lower k_cat values than the native enzyme indicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification. The chemical modification of xylanase from alkalothermophilic Bacillus revealed the presence of tryptophans in the active site (Dehspande, V, Hinge, J. and Rao, M. (1990) Biochim. Biophys. Acta 1041, 172–177). This finding and present studies demonstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from alkalothermophilic Bacillus sp.     

Effect of salts and Triton X-100 on ultrafiltration purification and properties of extracellular glucose oxidase from <Emphasis Type="Italic">Penicillium adametzii</Emphasis> LF F-2044.1

We compared the effectiveness of glucose oxidase isolation from the culture fluid of Penicillium adametzii LF F-2044.1 in the presence of ammonium sulfate, ammonium chloride, and Triton X-100. Ammonium chloride inhibited glucose oxidase in the culture fluid. This compound increased K _M (by 1.2–1.3 times), but decreased V _max for D-glucose oxidation (by 1.7–1.8 times). Ammonium sulfate had little effect on kinetic parameters. Combined treatment with salts and Triton X-100 was followed by a significant increase in the effectiveness of ultrafiltration purification of the culture fluid. The samples of glucose oxidase were electrophoretically characterized. The dependence of kinetic parameters on glucose oxidase concentration during oxidation of D-glucose was evaluated. The catalytic constant and k _cat/K _M ratio for glucose oxidase samples from the culture fluid isolated in the presence of additives significantly surpassed those for enzyme samples, which were obtained by ultrafiltration of the culture fluid with no additives and chromatography on aluminum oxide. The activity of glucose oxidase isolated from the culture fluid in the presence of ammonium chloride was lower compared to that of the enzyme obtained in the presence of ammonium sulfate. This agent is preferable for ultrafiltration of the culture fluid.     

Structure of the two-subsite beta-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane

The three-dimensional structure of the catalytically efficient β-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3 Å resolution). Most H bonds between inhibitor and protein occur within subsite −1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. E186 (pK_a 7.2) serves as catalytic acid. The pH (6-10) profile for is bell-shaped with pK_a’s 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.9 on the basic limb assigned to inhibitor. Mutation K99A eliminates pK_a 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14⁻E186⁻. A sedimentation equilibrium experiment estimates a K_d ([dimer]²/[tetramer]) of 7 × 10⁻⁹ M. Similar k_cat and k_cat/K_m values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms.     

Solvent isotope effects on α-glucosidase

The solvent kinetic isotope effects (SKIE) on the yeast α-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 °C. With p-nitrophenyl-d-glucopyranoside (pNPG), the dependence of k_cat/K_m on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, ^DOD(k_cat/K_m), of 1.9 (±0.3). The two pK_as characterizing the pH profile were increased in D₂O. The shift in pK_a2 of 0.6 units is typical of acids of comparable acidity (pK_a=6.5), but the increase in pK_a1 (=5.7) of 0.1 unit in going from H₂O to D₂O is unusually small. The initial velocities show substrate inhibition (K_is/K_m～200) with a small solvent isotope effect on the inhibition constant [^DODK_is=1.1 (±0.2)]. The solvent equilibrium isotope effects on the K_is for the competitive inhibitors d-glucose and α-methyl d-glucoside are somewhat higher [^DODK_i=1.5 (±0.1)]. Methyl glucoside is much less reactive than pNPG, with k_cat 230 times lower and k_cat/K_m 5×10⁴ times lower. The solvent isotope effect on k_cat for this substrate [=1.11 (±0. 02)] is lower than that for pNPG [=1.67 (±0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.     

The pH dependence of peptide hydrolysis by nitrocarboxypeptidase A

Hydrolysis of benzyloxycarbonyl-GlyGlyPhe by nitro(Tyr 248)carboxypeptidase A over the pH range 4.88–8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of K_m is scarcely affected. The peptidase activity has a pH dependence characterized by the following parameters: pK_E1 of 6.37 ± 0.19 and pK_E2 of 6.60 ± 0.17 in $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, and apparent pK of 5.59 ± 0.06 in K_cat. A spectroscopic pK of 6.75 ± 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pK_E2 in the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ profile, which appears to be shifted from a higher value, pK_E2 of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the k_cat profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, based on chemical modification and crystallographic evidence for the enzyme, is described.     

OptZyme: Computational Enzyme Redesign Using Transition State Analogues

OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., k_cat or k_cat/K_M) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using Escherichia coli β-glucuronidase as a benchmark system, we confirm that K_M correlates (R² = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, k_cat/K_M correlates (R² = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and k_cat correlates (R² = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved K_M, k_cat, and k_cat/K_M for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving K_M, k_cat, or k_cat/K_M. Mutants predicted to enhance the activity for para-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely H162S, L361G, W549R, and N550S.     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.     

Cloning, purification, and characterization of galactomannan-degrading enzymes from Myceliophthora thermophila

Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K _m and k _cat values are 0.4 mM and 15 sec^?1 for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galac-tomannans (GM) of various structures. The K _m and k _cat values are 1.3 mg/ml and 67 sec^?1 for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K _m and k _cat values are 0.08 mM and 35 sec^?1 for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.     

Evaluation of kinetic data: What the numbers tell us about PRMTs

Protein arginine N-methyltransferase (PRMT) kinetic parameters have been catalogued over the past fifteen years for eight of the nine mammalian enzyme family members. Like the majority of methyltransferases, these enzymes employ the highly ubiquitous cofactor S-adenosyl-l-methionine as a co-substrate to methylate arginine residues in peptidic substrates with an approximately 4-μM median K_M. The median values for PRMT turnover number (k_cat) and catalytic efficiency (k_cat/K_M) are 0.0051 s⁻¹ and 708 M⁻¹ s⁻¹, respectively. When comparing PRMT metrics to entries found in the BRENDA database, we find that while PRMTs exhibit high substrate affinity relative to other enzyme-substrate pairs, PRMTs display largely lower k_cat and k_cat/K_M values. We observe that kinetic parameters for PRMTs and arginine demethylase activity from dual-functioning lysine demethylases are statistically similar, paralleling what the broader enzyme families in which they belong reveal, and adding to the evidence in support of arginine methylation reversibility.     

Identification, Purification, and Characterization of Iminodiacetate Oxidase from the EDTA-Degrading Bacterium BNC1

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O₂. The temperature and pH optima for IDA oxidation were 35°C and 8, respectively. The apparent K_m for IDA was 4.0 mM with a k_cat of 5.3 s⁻¹. When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.     

Kinetic studies of Gly28:Ser mutant form of Bacillus pumilus lipase: Changes in kcat and thermal dependence

Lipases are useful catalysts for a wide variety of industrial purposes. Herein we report the stability and thermal dependence of the activity of wild-type Bacillus pumilus lipase (BplA) and four site-directed mutants designed to improve its thermal stability. The Gly28:Ser mutation produces a dramatic four-fold increase in its k_cat and a remarkable increase in its stability. While the increase in k_cat is temperature-independent, the increase in stability shows that the resultant interactions of this mutation have a strong enthalpic component. Thermal dependence of stability, k_cat, K_M and k_cat/K_M were analysed to gain insight on the structural effects of mutations on BplA. Our results are consistent with a gain in enzyme mobility for those mutants displaying enhanced catalytic properties; the analysis of thermal dependence of kinetic parameters indicates that the mutations did not change either the catalytic mechanism or the rate-limiting step of catalysis.     

Tissue kallikrein processes small proenkephalin peptides

Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin. Met⁵-Lys⁶-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37°C and pH 8.5, the K_M values for formation of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin were 129 and 191 μM, respectively. Corresponding k_cat values were 0.001 and 0.03 s⁻¹ and k_cat/K_M ratios were 8 and 1.6·10² M⁻¹ · s⁻¹, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met⁵-Arg⁶-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, K_M was 64 μM, k_cat was 4.5 s⁻¹, and the k_cat/K_M ratio was 7 · 10⁴ M⁻¹ · s⁻¹. At 5.5, the pH of the secretory granules, K_M, k_cat and k_cat/K_M were 184 μM, 1.9 s⁻¹ and 10⁴ M⁻¹ · s⁻¹, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds.