pK values for active site residues of carboxypeptidase A

The phenolic group of active site residue Tyr-248 in carboxypeptidase A has a pKa value of 10.06, as determined from the pH dependence of its rate of nitration by tetranitromethane. The decrease in enzyme activity (kcat/Km) in alkaline solution, characterized by a pKa value of approximately 9.0 (for cobalt carboxypeptidase A), is associated with the protonation state of an imidazole ligand of the active-site metal ion, as indicated by a selective pH dependence of the 1H NMR spectrum of the enzyme. Inhibition of the cobalt-substituted enzyme by 2-(1-carboxy-2-phenylethyl)phenol and its 4,6-dichloro- and 4-phenylazo-derivatives confirms that the decrease in enzyme activity (kcat/Km) in acidic solution, characterized by a pKa value of 5.8, is due to the protonation state of a water molecule bound to the active-site metal ion in the absence of substrate. Changes in the coordination number of the active-site metal ion are seen in its visible absorption spectrum as a consequence of binding of the phenolic inhibitors. Conventional concepts regarding the mechanisms of the enzyme are brought into question.     

Effects of two ionizing groups on the active site of human carbonic anhydrase B.

Investigation of some pH-dependent properties of human erythrocyte carbonic anhydrase B indicate that the active site is influenced by at least two charged groups. The properties studied include the pH dependence of inhibition of native, monocarboxamidomethyl, and monocarboxymethyl enzymes by iodide ion and the pH dependence of the visible spectra of the cobalt derivatives of these enzymes. One ionizing group has a pKa of about 7.3 in the native enzyme, 8.2 in the carboxyamidomethyl enzyme, and 9.0 in the carboxymethyl enzyme. It has a major influence on activity and anion inhibition, and on the visible spectra of the cobalt enzymes. A second group has a pKa of about 6.1 in native and modified enzymes. When zinc is at the active site, the secondary group in its acidic form decreases the Ki for I-. With the carboxyamidomethyl and carboxymethyl enzymes, the Ki decreases by about an order of magnitude. However, if cobalt is substituted for zinc in the modified enzymes, this group does not influence the Ki for I- and the binding of I- does not influence the pKa of the spectral transitions caused by ionization of this secondary group. In the case of nonalkylated Co2+-enzyme, another ionizing group with a pK of about 6.2 prevents the binging of I- at low pH. These results show that the active site is altered when cobalt is substituted for zinc in carbonic anhydrase B.     

pH effects in plasmin-catalysed hydrolysis of alpha-N-benzoyl-L-arginine compounds.

The steady-state kinetics of plasmin (EC 3.4.21.7) catalysed reactions with some alpha-N-benzoyl-L-arginine compounds is investigated in the pH range 5.8--9.0. The results are interpreted in terms of a three-step mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. Alpha-N-Benzoyl-L-arginine methyl ester and ethyl ester show the same pH behaviour. The kinetic parameter kc/Km is influenced by two groups with pK values of 6.5 and 8.4, respectively. kc is affected only by the group with pK equal to 6.5 and Km only by the group with pK equal to 8.4. It is suggested that the group with pK equal to 6.5 is the 1-chloro-3-tosyl-amido-7-amino-2-heptanone-sensitive histidine residue in the active site and that the group with pK equal to 8.4 is perhaps the alpha-amino group of the N-terminus in analogy to trypsin and chymotrypsin. alpha-N-Benzoyl-L-arginine amide is not hydrolysed by plasmin, but proves to be a competitive inhibitor, Ki = 12.8 +/- 1.8 mM, pH = 7.8. Also the product alpha-N-benzoyl-L-arginine is a competitive inhibitor, Ki = 26 +/- 3.1 mM, pH = 7.8. Estimates of individual rate constants are compared with similar trypsin data.     

Interaction of carboxypeptidase A with carbamate and carbonate esters

The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine binds well to carboxypeptidase A in the manner of peptide substrates. The ester exhibits linear competitive inhibition toward carboxypeptidase A catalyzed hydrolysis of the amide hippuryl-L-phenylalanine (Ki = 1.0 X 10(-3) M at pH 7.5) and linear noncompetitive inhibition toward hydrolysis of the specific ester substrate O-hippuryl-L-beta-phenyllactate (Ki = 1.4 X 10(-3) M at pH 7.5). Linear inhibition shows that only one molecule of inhibitor is bound per active site at pH 7.5. The hydrolysis of the carbamate ester is not affected by the presence of 10(-8)-10(-9) M enzyme (the concentrations employed in inhibition experiments), but at an enzyme concentration of 3 X 10(-6) M catalysis can be detected. The value of kcat at 30 degrees C, mu = 0.5 M, and pH 7.45 is 0.25 s-1, and Km is 1.5 X 10(-3) M. The near identity of Km and Ki shows that Km is a dissociation constant. Substrate inhibition can be detected at pH less than 7 but not at pH values above 7, which suggests that a conformational change is occurring near that pH. The analogous carbonate ester O-(phenoxycarbonyl)-L-beta-phenyllactic acid is also a substrate for the enzyme. The Km is pH independent from pH 6.5 to 9 and has the value of 7.6 X 10(-5) M in that pH region. The rate constant kcat is pH independent from pH 8 to 10 at 30 degrees C (mu = 0.5 M) with a limiting value of 1.60 s-1. Modification of the carboxyl group of glutamic acid-270 to the methoxyamide strongly inhibits the hydrolysis of O-(phenoxycarbonyl)-L-beta-phenyllactic acid. Binding of beta-phenyllactate esters and phenylalanine amides must occur in different subsites, but the ratios of kcat and kcat/Km for the structural change from hippuryl to phenoxy in each series are closely similar, which suggests that the rate-determining steps are mechanistically similar.     

Acid-base chemical mechanism of homocitrate synthase from Saccharomyces cerevisiae

Homocitrate synthase (acetyl-coenzyme A:2-ketoglutarate C-transferase; E.C. 2.3.3.14) catalyzes the condensation of AcCoA and alpha-ketoglutarate to give homocitrate and CoA. The enzyme was found to be a Zn-containing metalloenzyme using inductively coupled plasma mass spectrometry. Dead-end analogues of alpha-ketoglutarate were used to obtain information on the topography of the alpha-ketoglutarate binding site. The alpha-carboxylate and alpha-oxo groups of alpha-ketoglutarate are required for optimum binding to coordinate to the active site Zn. Optimum positioning of the alpha-carboxylate, alpha-oxo, and gamma-carboxylate of alpha-ketoglutarate is likely mimicked by the location in space of the 2-carboxylate, pyridine nitrogen, and 4 carboxylate of pyridine 2,4-dicarboxylate. The pH dependence of the kinetic parameters was determined to obtain information on the chemical mechanism of homocitrate synthase. The V profile is bell shaped with slopes of 1 and -1, giving pKa values of 6.7 and 8.0, while V/K(AcCoA) exhibits a slope of 2 on the acidic side with an average pKa value of 6.6 and a slope of -2 on basic side of the profile with an average pKa value of 8.2. The V/K(alpha-Kg) pH-rate profile exhibits a single pKa of 6.9 on the acidic side and two on the basic side with an average value of 7.8. The pH dependence of the Ki for glyoxylate, a competitive inhibitor vs alpha-ketoglutarate, gives a pKa of 7.1 for a group, required to be protonated for optimum binding. Data suggest a chemical mechanism for the enzyme in which alpha-ketoglutarate first binds to the active site Zn via its alpha-carboxylate and alpha-oxo groups, followed by acetyl-CoA. A general base then accepts a proton from the methyl of acetyl-CoA, and a general acid protonates the carbonyl of alpha-ketoglutarate in the formation of homocitryl-CoA. The general acid then acts as a base in deprotonating Zn-OH2 in the hydrolysis of homocitryl-CoA to give homocitrate and CoA. A solvent deuterium kinetic isotope effect of 1 is measured for homocitrate synthase, while a small pH-independent primary kinetic deuterium isotope effect (approximately 1.3) is observed using deuterioacetyl-CoA. Data suggest rate-limiting condensation to form the alkoxide of homocitryl-CoA, followed by hydrolysis to give products.     

pH dependency of the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli

The variation with pH of the kinetic parameters associated with the mutase and dehydrogenase reactions catalyzed by chorismate mutase-prephenate dehydrogenase has been determined with the aim of elucidating the role that ionizing amino acid residues play in binding and catalysis. The pH dependency of log V for the dehydrogenase reaction shows that the enzyme possesses a single ionizing group with a pK value of 6.5 that must be unprotonated for catalysis. This same group is observed in the V/Kprephenate, as well as in the V/KNAD, profile. The V/Kprephenate profile exhibits a second ionizing residue with a pK value of 8.4 that must be protonated for the binding of prephenate to the enzyme. For the mutase reaction, the V/Kchorismate profile indicates the presence of three ionizing residues at the active site. Two of these residues, with similar pK values of about 7, must be protonated, while the third, with a pK value of 6.3, must be unprotonated. It can be concluded that all three groups are concerned with the binding of chorismate to the enzyme since the maximum velocity of the mutase reaction is essentially independent of pH. This conclusion is confirmed by the finding that the Ki profile for the competitive inhibitor, (3-endo,8-exo)-8-hydroxy-2-oxabicyclo[3.3]non-6-ene-3,5-dicarboxylic acid, shows the same three ionizing groups as observed in the V/Kchorismate profile. By contrast, the Ki profile for carboxyethyldihydrobenzoate shows only one residue, with a pK value of 7.3, that must be protonated for binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue factor alters the pK(a) values of catalytically important factor VIIa residues

Blood coagulation is triggered when the serine protease factor VIIa (fVIIa) binds to cell surface tissue factor (TF) to form the active enzyme-cofactor complex. TF binding to fVIIa allosterically augments the enzymatic activity of fVIIa toward macromolecular substrates and small peptidyl substrates. The mechanism of this enhancement remains unclear. Our previous studies have indicated that soluble TF (sTF; residues 1-219) alters the pH dependence of fVIIa amidolytic activity (Neuenschwander et al. (1993) Thromb. Haemostasis 70, 970), indicating an effect of TF on critical ionizations within the fVIIa active center. The pKa values and identities of these ionizable groups are unknown. To gain additional insight into this effect, we have performed a detailed study of the pH dependence of fVIIa amidolytic activity. Kinetic constants of Chromozym t-PA (MeSO(2)-D-Phe-Gly-Arg-pNA) hydrolysis at various pH values were determined for fVIIa alone and in complex with sTF. The pH dependence of both enzymes was adequately represented using a diprotic model. For fVIIa alone, two ionizations were observed in the free enzyme (pK(E1) = 7.46 and pK(E2) = 8.67), with at least a single ionization apparent in the Michaelis complex (pK(ES1) similar 7.62). For the fVIIa-TF complex, the pK(a) of one of the two important ionizations in the free enzyme was shifted to a more basic value (pK(E1) = 7.57 and pK(E2) = 9.27), and the ionization in the Michaelis complex was possibly shifted to a more acidic pH (pK(ES1) = 6.93). When these results are compared to those obtained for other well-studied serine proteases, K(E1) and K(ES1) are presumed to represent the ionization of the overall catalytic triad in the absence and presence of substrate, respectively, while K(E2) is presumed to represent ionization of the alpha-amino group of Ile(153). Taken together, these results would suggest that sTF binding to fVIIa alters the chemical environment of the fVIIa active site by protecting Ile(153) from deprotonation in the free enzyme while deprotecting the catalytic triad as a whole when in the Michaelis complex.     

Quantitative structure-activity relationships for the pre-steady state acetylcholinesterase inhibition by carbamates

4-Nitrophenyl-N-substituted carbamates (1) are characterized as pseudosubstrate inhibitors of acetylcholinesterase. The first step is formation of the enzyme-inhibitor tetrahedral intermediate with the inhibition constant (Ki), the second step is formation of the carbamyl enzyme with the carbamylation constant (kc), and the third step is hydrolysis of the carbamyl enzyme with decarbamylation constant (kd). According to pre-steady state kinetics the Ki step is divided further into two steps: (1) formation of the enzyme-inhibitor complex with the dissociation constant (KS) and (2) formation of the enzyme-inhibitor tetrahedral intermediate from the complex with the equilibrium constant (k2/k-2). Since the inhibitors are protonated in pH 7.0 buffer solution, the virtual dissociation constant (KS') of the enzyme-protonated inhibitor complex can be calculated from the equation, -log KS'=-log KS-pKa + 14. The -logKS, -log KS', log k2, and log k-2 values are multiply linearly correlated with the Jave equation (log(k/k0)=rho*sigma* + deltaEs + psi pi). For -log KS'-sigma*-Es)pi-correlation, the rho* value of -0.4 indicates that the enzyme-protonated inhibitor complexes have more positive charges than the protonated inhibitors, the delta value of 0.44 suggests that the bulkily substituted inhibitors lessen the reaction due to the difficulty of the inhibitors to enter the narrow enzyme active site gorge, and the psi value of 0.27 implies that the inhibitors with hydrophobic substituents accelerate the inhibitors entering the active site gorge of the enzyme. For log k2/k-2,-sigma*-Es-pi-correlation, the rho* value of 1.1 indicates that the enzyme-protonated inhibitor tetrahedral intermediates have more negative charges than the enzyme-protonated inhibitor complexes, the delta value of 0.15 suggests that the bulkily substituted inhibitors are difficult to bind into a small acyl binding site of the enzyme, and the psi value of -0.3 implies that the inhibitors with hydrophobic substituents resist binding to the hydrophilic acyl binding site of the enzyme.     

A new class of potent reversible inhibitors of metallo-proteinases: C-terminal thiol-peptides as zinc-coordinating ligands

A number of substrate analogous peptides containing a phosphoramidate, phosphonate ester, hydroxamate, carboxylate or sulfhydryl group are known to be inhibitors of thermolysin and other metalloproteinases. According to the specificity, most of the inhibitors mimic the prime site of the active center. Hitherto, peptidyl derivatives with a thiol group at the C-terminus have not been described. We have synthesized the protected cysteamides Ac-Ala-Ala-CA-SH and Z-Aa1-Aa2-CA-SH (Aa1: Ala, Pro; Aa2: Ala, Leu). The binding of these thiol peptide inhibitors to the metalloproteinases is characterized first by the coordination of the thiolate group of the inhibitor to the catalytic zinc ion and second by the subsite interaction of the peptide ligand in the active site of the enzyme. All peptide derivatives were competitive inhibitors of the zinc metalloproteinase thermolysin. The strongest inhibition was found with Z-Pro-Leu-CA-SH (Ki = 30 microM). Substitution of the N-protecting benzyloxycarbonyl residue towards the acetyl group in the peptide inhibitor, the inhibition constant decreased about 25 times.     

Binding of reversible spin-labeled inhibitors with an butyrylcholinesterase active center

The interaction of spin-labeled metacyn, procaine, carbolin and bivalent cations (Ca2+, Co2+, Ni2+) with butyrylcholinesterase (BChE) was studied by ESR and enzyme kinetic methods. The effect of pH, ionic strength and organic solvent was analysed. Spin-labeled metacyn binds at the anionic site of BChE active centre. This complex is stabilized both with coulombic and hydrophobic interactions, ionizing group of active centre with pK 6-7 also affects the binding. Spin-labeled procaine appeared to be enzyme competitive inhibitor (Ki = 4 X 10(-5) M) and is located, most probably, at the same site. Activating effect of Ca2+ ions on BChE was confirmed. Simultaneous application of spin labels and paramagnetic ions demonstrates that cations Co2+ and Ni2+ bind with BChE in the close vicinity of spin-labeled inhibitor site. Paramagnetic cations are located more closely to the cationic part of the inhibitor molecule than to the hydrophobic one, and can be displaced by surplus of Ca2+ ions. The experimental data testify the model of anionic centre which consists of bivalent metal ions and aminoalcyl cationic group subsites and is located in a hydrophobic pocket of the enzyme surface.     

Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a pK of 7.2 +/- 0.1. By analogy to other serine proteases this pK is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D2Okcat/Km=2.9+/-0.2 and a D2Okcat=1.7+/-0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A "burst" of product release during pre steady-state Gly-Pro-pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (approximately 15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly (kon) depends upon the ionization of an enzyme residue with a pK of 6.2 +/- 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a pK of 7.9 +/- 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme-inhibitor complex was reversible and dissociated with a koff of (5.5 +/- 0.4) x 10(-5) s(-1), thus yielding a Ki* (as koff/kon) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors (phi) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme-inhibitor complex.     

Leaving group dependence in the phosphorylation of Escherichia coli alkaline phosphatase by monophosphate esters

Values of kcat and Km have been measured for the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 18 aryl and 12 alkyl monophosphate esters at pH 8.00 and 25 degrees C. A Br?nsted plot of log (kcat/Km) (M-1 s-1) vs. the pK of the leaving hydroxyl group exhibits two regression lines: log (kcat/Km) = -0.19 (+/- 0.02) pKArOH + 8.14 (+/- 0.15) log (kcat/Km) = -0.19 (+/- 0.01) pKROH + 5.89 (+/- 0.17) Alkyl phosphates with aryl or large lipophilic side chains are not correlated by the above equations and occupy positions intermediate between the two lines. The observed change in effective charge on the leaving oxygen of the ester (-0.2) is very small, consistent with substantial electrophilic participation of the enzyme with this atom. Cyclohexylammonium ion is a noncompetitive inhibitor against 4-nitrophenyl phosphate substrate at pH 8.00, and neutral phenol is a competitive inhibitor (Ki = 82.6 mM); these data and the 100-fold larger reactivity of aryl over alkyl esters are consistent with the existence of a lipophilic binding site for the leaving group of the substrate. The absence of a major steric effect in kcat/Km for substituted aryl esters confirms that the leaving group in the enzyme--substrate complex points away from the surface of the enzyme. Arguments are advanced to exclude a dissociative mechanism (involving a metaphosphate ion) for the enzyme-catalyzed substitution at phosphorus.     

Spectroscopic and kinetic evidence for the thiolate anion of glutathione at the active site of glutathione S-transferase

Ultraviolet difference spectroscopy of the binary complex of isozyme 4-4 of rat liver glutathione S-transferase with glutathione (GSH) and the enzyme alone or as the binary complex with the oxygen analogue, gamma-L-glutamyl-L-serylglycine (GOH), at neutral pH reveals an absorption band at 239 nm (epsilon = 5200 M-1 cm-1) that is assigned to the thiolate anion (GS-) of the bound tripeptide. Titration of this difference absorption band over the pH range 5-8 indicates that the thiol of enzyme-bound GSH has a pKa = 6.6, which is about 2.4 pK units less than that in aqueous solution and consistent with the kinetically determined pKa previously reported [Chen et al. (1988) Biochemistry 27, 647]. The observed shift in the pKa between enzyme-bound and free GSH suggests that about 3.3 kcal/mol of the intrinsic binding energy of the peptide is utilized to lower the pKa into the physiological pH range. Apparent dissociation constants for both GSH and GOH are comparable and vary by a factor of less than 2 over the same pH range. Site occupancy data and spectral band intensity reveal large extinction coefficients at 239 nm (epsilon = 5200 M-1 cm-1) and 250 nm (epsilon = 1100 M-1 cm-1) that are consistent with the existence of either a glutathione thiolate (E.GS-) or ion-paired thiolate (EH+.GS-) in the active site. The observation that GS- is likely the predominant tripeptide species bound at the active site suggested that the carboxylate analogue of GSH, gamma-L-glutamyl-(D,L-2-aminomalonyl)glycine, should bind more tightly than GSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH dependence of the inhibition of chymotrypsin by a peptidyl trifluoromethyl ketone

The effects of pH on the kinetics of association and dissociation of chymotrypsin and the dipeptidyl trifluoromethyl ketone (TFK) N-acetyl-L-leucyl-L-phenylalanyltrifluoromethane (1) were examined through the pH range 4-9.5. The pH dependence of the association rate (kon) is similar to that of kcat/Km for ester and peptide substrates and is dependent on two pK's at 7.0 and 8.9. We assign these pK's to the active site His and to the amino group of the N-terminal isoleucine residue. Ki for the complex of 1 and chymotrypsin has a pH dependence very similar to that of kon, and we conclude that the same ionizable groups which determine the pH dependence of kon are involved. The dissociation constant of the enzyme-inhibitor complex (koff) shows no pH dependence between pH 4 and pH 9.5. The data indicate that the inhibitor reacts with a form of the enzyme in which His 57 is unprotonated, and the resulting complex contains no groups which ionize between pH 4 and pH 9.5. This is consistent with conclusions previously reached from NMR data (Liang & Abeles, 1987). These experiments led to the conclusion that 1 reacts with chymotrypsin to form a tetrahedral complex in which His 57 is protonated (pK greater than 9.5) and the OH group of serine 195 has added to the carbonyl group of 1 to form an ionized hemiketal (pK less than 4.9). The pK of His 57 is increased by greater than 3 units over that in the free enzyme, and the pK of the hemiketal decreased by greater than 4 units compared to the pK in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extreme pKa displacements at the active sites of FMN-dependent alpha-hydroxy acid-oxidizing enzymes.

Flavocytochrome b2 (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related alpha-hydroxy acid-oxidizing FMN-dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate alpha-proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pKa belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the alpha-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.     

Chemical mechanism of the adenosine cyclic 3',5'-monophosphate dependent protein kinase from pH studies

The pH dependence of kinetic parameters and inhibitor dissociation constants for the adenosine cyclic 3',5'-monophosphate dependent protein kinase reaction has been determined. Data are consistent with a mechanism in which reactants selectively bind to enzyme with the catalytic base unprotonated and an enzyme group required protonated for peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) binding. Binding of the peptide apparently locks both of the above enzyme residues in their correct protonation state. MgATP preferentially binds fully ionized and requires an enzyme residue (probably lysine) to be protonated. The maximum velocity and V/KMgATP are pH independent. The V/K for Ser-peptide is bell-shaped with pK values of 6.2 and 8.5 estimated. The pH dependence of 1/Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly is also bell-shaped, giving pK values identical with those obtained for V/KSer-peptide, while the Ki for MgAMP-PCP increases from a constant value of 650 microM above pH 8 to a constant value of 4 mM below pH 5.5. The Ki for uncomplexed Mg2+ obtained from the Mg2+ dependence of V and V/KMgATP is apparently pH independent.     

Interaction of Trimeresurus flavoviridis phospholipase A2 and its fragment with calcium ion

Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.     

Mechanism of action of isopentenyl pyrophosphate isomerase: evidence for a carbonium ion intermediate

Isopentenyl pyrophosphate isomerase catalyzes the interconversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The isomerase from yeast has been purified to near homogeneity (purity greater than 90%). The substrate analogue (Z)-3-(trifluoromethyl)-2-butenyl pyrophosphate reacts at less than 1.8 X 10(-6) times the rate of dimethylallyl pyrophosphate. The enzyme is irreversibly inactivated by 2-(dimethyl-amino)ethyl pyrophosphate (I). These observations are consistent with a carbonium ion mechanism for the isomerization. Compound I is an analogue of the intermediate carbonium ion and probably acts as a transition state analogue. For I, kon' = 2.1 X 10(6) M-1 min-1. No off-rate was detected and, therefore, Ki less than 1.4 X 10(-11) M. Upon denaturation of the inactivated enzyme, I is released unchanged. 2-(Trimethylammonio)ethyl pyrophosphate also inhibits with Ki' = 7 X 10(-7) M, kon' = 4.4 X 10(4) M-1 min-1, and koff = 0.03 min-1. Substrate analogues without a positively charged nitrogen were relatively poor inhibitors. The best inhibitor of these is ethyl pyrophosphate, Ki = 10(-4) M. The enzyme is inactivated by sulfhydryl-selective reagents. These reagents also prevent binding of I to the enzyme. The inactivation by iodoacetamide is dependent upon one ionizable group (pK = 9.3). The pH dependence of V and V/K for the isomerase-catalyzed reaction also depends upon a group with pK = 9.3.     

QSAR for phospholipase A2 inhibitions by 1-acyloxy-3-N-n-octylcarbamyl-benzenes

1-Acyloxy-3-N-n-octylcarbamyl-benzenes are potent reversible competitive inhibitors of Naja mocambique mocambique phospholipase A2 with the Ki values from 9.6 to 119 microM. The pKi values are correlated to both Taft substituent constant sigma* and Hansch hydrophobicity constant pi. The pre-steady state inhibition studies indicate that the pK(S) values for the first inhibition step are linearly correlated to sigma* alone with the rho* of -0.09 for this correlation. Thus, the first inhibition step may involve the insertion of the inhibitor to hepta-coordinated Ca2+ ion of the enzyme to form the octa-coordinated Ca2+ ion of the enzyme. The log(k2/k(-2)) values for the second inhibition step are linearly correlated to pi alone, and the psi value for this correlation is 0.13. Therefore, the second step inhibition step may involve the van der Waals' interaction between the acyl group of the inhibitor and Tyr 69 of the enzyme.     

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.