Fractionation factors and activation energies for exchange of the low barrier hydrogen bonding proton in peptidyl trifluoromethyl ketone complexes of chymotrypsin

NMR investigations have been carried out of complexes between bovine chymotrypsin Aalpha and a series of four peptidyl trifluoromethyl ketones, listed here in order of increasing affinity for chymotrypsin: N-Acetyl-L-Phe-CF3, N-Acetyl-Gly-L-Phe-CF3, N-Acetyl-L-Val-L-Phe-CF3, and N-Acetyl-L-Leu-L-Phe-CF3. The D/H fractionation factors (phi) for the hydrogen in the H-bond between His 57 and Asp 102 (His 57-Hdelta1) in these four complexes at 5 degreesC were in the range phi = 0.32-0.43, expected for a low-barrier hydrogen bond. For this series of complexes, measurements also were made of the chemical shifts of His 57-Hepsilon1 (delta2,2-dimethylsilapentane-5-sulfonic acid 8.97-9. 18), the exchange rate of the His 57-Hdelta1 proton with bulk water protons (284-12.4 s-1), and the activation enthalpies for this hydrogen exchange (14.7-19.4 kcal.mol-1). It was found that the previously noted correlations between the inhibition constants (Ki 170-1.2 microM) and the chemical shifts of His 57-Hdelta1 (delta2, 2-dimethylsilapentane-5-sulfonic acid 18.61-18.95) for this series of peptidyl trifluoromethyl ketones with chymotrypsin [Lin, J., Cassidy, C. S. & Frey, P. A. (1998) Biochemistry 37, 11940-11948] could be extended to include the fractionation factors, hydrogen exchange rates, and hydrogen exchange activation enthalpies. The results support the proposal of low barrier hydrogen bond-facilitated general base catalysis in the addition of Ser 195 to the peptidyl carbonyl group of substrates in the mechanism of chymotrypsin-catalyzed peptide hydrolysis. Trends in the enthalpies for hydrogen exchange and the fractionation factors are consistent with a strong, double-minimum or single-well potential hydrogen bond in the strongest complexes. The lifetimes of His 57-Hdelta1, which is solvent shielded in these complexes, track the strength of the hydrogen bond. Because these lifetimes are orders of magnitude shorter than those of the complexes themselves, the enzyme must have a pathway for hydrogen exchange at this site that is independent of dissociation of the complexes.     

Roles of histidine 752 and glutamate 699 in the pH dependence of mouse band 3 protein-mediated anion transport

In the accompanying paper we have shown that four different histidine residues are involved in the maintenance of mouse band 3 in a state in which it is able to execute its anion transport function. Here we focus on the functional significance of His 752 and demonstrate that this residue, together with Glu 699, plays a key role in the control of pH dependence of Cl- transport. Mouse band 3-encoding cRNA was expressed in Xenopus oocytes, and band 3-mediated Cl- transport was measured at zero membrane potential over the pH range 6.0-9.2. Transport decreased with increasing H+ concentration and was governed by a single pK of 5.8. After correction for temperature differences, this result agrees well with measurements in erythrocyte ghosts of Cl- flux by Funder and Wieth [Funder, J., & Wieth, J. O. (1976) J. Physiol. 262, 679-698] and our own determinations by 35Cl NMR spectroscopy of Cl- exchange between the substrate binding site and the medium. After mutation of either Glu 699 to Asp or of His 752 to Ser, the maximal rate of transport is reduced and the rate of anion exchange is now governed by a single pK of about 6.8-6.9. This suggests that the formation of a hydrogen bond between His 752 and Glu 699 is essential for the decrease of band 3-mediated Cl- transport at low pH. We suggest that in the wild type band 3 both the decrease of the chloride exchange between the medium and the substrate binding site and the inhibition of chloride translocation across the membrane are dominated by a common rate-limiting step and that this step involves hydrogen bond formation between Glu 699 and His 752.     

Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.     

Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme. For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena. First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J. D., et al. (1994) Eur. J. Biochem. 244, 81-87], for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN. Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1. The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.     

Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway

A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem. 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.     

Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation

Ribonucleotide reductases (RNRs) catalyze the rate-determining step in DNA biosynthesis: conversion of nucleotides to deoxynucleotides. The RNR from Lactobacillus leichmannii utilizes adenosylcobalamin (AdoCbl) as a cofactor and, in addition to nucleotide reduction, catalyzes the exchange of tritium from [5'-3H]-AdoCbl with solvent. Examination of this exchange reaction offers a unique opportunity to investigate the early stages in the nucleotide reduction process [Licht S. S., Gerfen, G. J., and Stubbe, J. (1996) Science 271, 477-481]. The kinetics of and requirements for this exchange reaction have been examined in detail. The turnover number for 3H washout is 0.3 s-1, and it requires an allosteric effector dGTP (Km = 17 +/- 3 microM), AdoCbl (Km = 60 +/- 9 microM) and no external reductant. The effects of active-site mutants of RTPR (C119S, C419S, C731S, C736S, and C408S) on the rate of the exchange reaction have been determined, and only C408 is essential for this process. The exchange reaction has previously been monitored by stopped-flow UV-vis spectroscopy, and cob(II)alamin was shown to be formed with a rate constant of 40 s-1 [Tamao, Y., and Blakley, R. L. (1973) Biochemistry 12, 24-34]. This rate constant has now been measured in D2O, with [5'-2H2]-AdoCbl in H2O, and with [5'-2H2]-AdoCbl in D2O. A comparison of these results with those for AdoCbl in H2O revealed kH/kD of 1.6, 1.7, and 2.7, respectively. The absolute amounts of cob(II)alamin generated with [5'-2H2]-AdoCbl in D2O in comparison with AdoCbl in H2O reveal twice as much cob(II)alamin in the former case. Similar transient kinetic studies with C408S RTPR reveal no cob(II)alamin formation. These experiments allow proposal of a minimal mechanism for this exchange reaction in which RNR catalyzes homolysis of the carbon-cobalt bond in a concerted fashion, to generate a thiyl radical on C408, cob(II)alamin, and 5'-deoxyadenosine.     

American Board of Emergency Medicine Longitudinal Study of Emergency Physicians

Mutant adenylosuccinate lyases of Bacillus subtilis were prepared by site-directed mutagenesis with replacements for His141, previously identified by affinity labeling as being in the active site [Lee, T. T., Worby, C., Dixon, J. E., and Colman, R. F. (1997) J. Biol. Chem. 272, 458-465]. Four substitutions (A, L, E, Q) yield mutant enzyme with no detectable catalytic activity, while the H141R mutant is about 10(-)5 as active as the wild-type enzyme. Kinetic studies show, for the H141R enzyme, a Km that is only 3 times that of the wild-type enzyme. Minimal activity was also observed for mutant enzymes with replacements for His68 [Lee, T. T., Worby, C., Bao, Z. -Q., Dixon, J. E., and Colman, R. F. (1998) Biochemistry 37, 8481-8489]. Measurement of the reversible binding of radioactive adenylosuccinate by inactive mutant enzymes with substitutions at either position 68 or 141 shows that their affinities for substrate are decreased by only 10-40-fold. These results suggest that His141, like His68, plays an important role in catalysis, but not in substrate binding. Evidence is consistent with the hypothesis that His141 and His68 function, respectively, as the catalytic base and acid. Circular dichroism spectroscopy and gel filtration chromatography conducted on wild-type and all His141 and His68 mutants reveal that none of the mutant enzymes exhibits major structural changes and that all the enzymes are tetramers. Mixing inactive His141 with inactive His68 mutant enzymes leads to striking increases in catalytic activity. This complementation of mutant enzymes indicates that His141 and His68 come from different subunits to form the active site. A tetrameric structure of adenylosuccinate lyase was constructed by homology modeling based on the known structures in the fumarase superfamily, including argininosuccinate lyase, delta-crystallin, fumarase, and aspartase. The model suggests that each active site is constituted by residues from three subunits, and that His141 and His68 come from two different subunits.     

The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates

The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.     

Mechanistic studies on the 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis

The reaction mechanism of 8-amino-7-oxopelargonate (8-amino-7-oxononoate) synthase from Bacillus sphaericus, an enzyme dependent on pyridoxal 5'-phosphate (pyridoxal-P), which catalyzes the condensation of L-alanine with pimeloyl-CoA, the second step of biotin biosynthesis, has been studied. To facilitate mechanistic studies, an improved over-expression system in Escherichia coli, and a new continuous spectrophotometric assay for 8-amino-7-oxopelargonate synthase were designed. In order to discriminate between the two plausible basic mechanisms that can be put forth for this enzyme, that is: (a) formation of the pyridoxal-P-stabilized carbanion by abstraction of the C2-H proton of the alanine-pyridoxal-P aldimine, followed by acylation and decarboxylation, and (b) formation of the carbanion by decarboxylation followed by acylation, the fate of the C2-H proton of alanine during the course of the reaction has been examined using 1H NMR. Spectra of the 8-amino-7-oxopelargonate formed using either L-[2-2H]alanine in H2O or L-alanine in D2O, showed that the C2-H proton of alanine is lost during the reaction and that the C8-H proton of 8-amino-7-oxopelargonate is derived from the solvent, a result that is only consistent with mechanism (a). Furthermore 8-amino-7-oxopelargonate synthase catalyzes, in the absence of pimeloyl-CoA, the stereospecific exchange, with retention of configuration, of the C2-H proton of L-alanine with the solvent protons. Similarly, 8-amino-7-oxopelargonate synthase catalyzes the exchange of the C8-H proton of 8-amino-7-oxopelargonate. In addition to these exchange reactions, 8-amino-7-oxopelargonate synthase catalyzes an abortive transamination yielding an inactive pyridoxamine 5'-phosphate (pyridoxamine-P) form of 8-amino-7-oxopelargonate synthase and pyruvate. Kinetic analysis gave a rate constant of kexch. = 1.8 min-1 for the exchange reaction which is 10 times lower than the catalytic constant and a rate constant of ktrans. = 0.11 h-1 for the transamination. Finally deuterium kinetic isotope effects (KIE) were measured at position 2 of L-alanine (DV = 1.3) and in D2O (D2OV = 4.0). The magnitudes of the KIE are consistent with a partially rate-limiting abstraction of the C2-H proton of alanine and a partially rate-limiting reprotonation step. Taken together, all these results show that 8-amino-7-oxopelargonate synthase utilizes mechanism (a). 8-Amino-7-oxopelargonate synthase and 5-aminolevulinate synthase, which has also been shown to use mechanism (a), belong to a class of pyridoxal-P-dependent enzymes that catalyze the formation of alpha-oxoamines. Based on the fact that all these alpha-oxoamine synthases share strong sequence similarities, we postulate that they also share the same reaction mechanism.     

Dissecting the contributions of a specific side-chain interaction to folding and catalysis of Bacillus stearothermophilus lactate dehydrogenase

X-ray crystallography predicts hydrogen-bonding interactions between the side chains of Thr198 and two other amino acid residues, Glu194 (adjacent to the catalytic His195) and Ser318 (on the alpha-H helix which rearranges on substrate binding). In order to investigate the contribution of this conserved amino acid residue, Thr198, two mutants of Bacillus stearothermophilus lactate dehydrogenase were created (Val198 and Ile198). The steady-state kinetic parameters for both mutant enzymes were very similar with increased substrate Km and reduced kcat when compared with the wild-type enzyme. The mutation Val198 allowed non-productive binding of pyruvate to the unprotonated form of His195. Steady-state kinetic parameters determined for the Val198 mutant enzyme in high solvent viscosity suggested both an altered rate-limiting step in catalysis and implicated Thr198 in allosteric activation by the effector fructose 1,6-bisphosphate (Fru1,6P2). A shift in the Fru1,6P2 activation constant for the Val198 mutant enzyme suggested that Thr198 stabilises the catalytically competent (Fru1,6P2-activated) form of the enzyme by 6.6 kJ/mol. However, Thr198 was not important for maintaining the thermal stability of the Fru1,6P2-activated form. Equilibrium unfolding in guanidinium chloride indicated that Thr198 contributes 17.2 kJ/mol subunits towards the tertiary structural stability. The results emphasise the importance of the side chain-hydroxyl group of Thr198 which is required for (a) productive substrate binding, (b) allosteric activation and (c) protein conformational stability. The characteristics of the B. stearothermophilus lactate dehydrogenase mutations reported here were significantly different from those of the same mutations made in the corresponding position of the analogous enzyme Thermus flavus malate dehydrogenase [Nishiyama, M., Shimada, K., Horinouchi, S., & Beppu, T. (1991) J. Biol. Chem. 266, 14294-14299].     

Properties of the His57-Asp102 dyad of rat trypsin D189S in the zymogen, activated enzyme, and alpha1-proteinase inhibitor complexed forms

Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, alpha1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp102 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human alpha1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and alpha-chymotrypsin. In these studies the pKa of His57 has been determined from the pH dependence of the 1H NMR signal from the Hdelta1 proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pKa = 7.8 +/- 0.1; Hill coefficient = 0. 86 +/- 0.05) decreased the pKa of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pKa = 6.7 +/- 0.1; Hill coefficient = 1.37 +/- 0.08). The binding of alpha1-proteinase inhibitor to trypsin D189S led to an increase in the pKa value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pKa = 8.1 +/- 0. 1; Hill coefficient = 0.70 +/- 0.08), as was observed for the zymogen. In spite of these differences in the pKa of His57 in the zymogen, active enzyme, and alpha1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 Hdelta1 proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the Hdelta1 proton of His57 was relatively sharp, at temperatures between 5 and 20 degrees C at both low pH (5.2) and high pH (10.0), in spectra of bovine alpha-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human alpha1-proteinase inhibitor; however, in spectra of the complex between alpha-chymotrypsin and human alpha1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5 degrees C).     

Systemic atherosclerosis in dogs: histopathological and immunohistochemical studies of atherosclerotic lesions

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 3,3-Dimethyl-6-(3-lauroylureido)-7-oxo-4-thia-1-azabicyclo[3,2,0] heptane-2-carboxylic acid (1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 72 microM). This beta-lactam did not inhibit other phospholipases, including the human nonpancreatic secreted phospholipase A2. The inhibition of cPLA2 was found not to be time-dependent. This, along with the observation that the degradation of the inhibitor was not catalyzed by the enzyme, demonstrates that the inhibition does not result from the formation of an acyl-enzyme intermediate with the active site serine residue. Moreover, the ring-opened form of 1 is also able to inhibit cPLA2 with near-equal potency. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol containing 6-10 mole percent of 1-palmitoyl-2-[1-14C]-arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition was defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.5 +/- 0.1 mole% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.4 +/- 0.1 mole%. Thus, 1 represents a novel structural class of inhibitors of cPLA2 which partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site.     

Substrate recognition by "password" in p-hydroxybenzoate hydroxylase

The flavin of p-hydroxybenzoate hydroxylase (PHBH) adopts two conformations [Gatti, D. L., Palfey, B. A., Lah, M.-S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science 266, 110-114; Schreuder, H. A., Mattevi, A., Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., and van Berkel, W. J. H. (1994) Biochemistry 33, 10161-10170]. Kinetic studies detected the movement of the flavin from the buried conformation to the exposed conformation caused by the binding of NADPH prior to its reaction with the flavin. The pH dependence of the rate constant for flavin reduction in wild-type PHBH and the His72Asn mutant indicates that the deprotonation of bound p-hydroxybenzoate is also required for flavin movement, and is accomplished by the same internal proton transport network previously found to be involved in substrate oxidation. The linkage of substrate deprotonation to flavin movement constitutes a novel mode of molecular recognition in which the enzyme tests the suitability of aromatic substrates before committing to the catalytic cycle.     

Hydrogen bond network in the distal site of peroxidases: spectroscopic properties of Asn70 --> Asp horseradish peroxidase mutant

The distal His in peroxidases forms a hydrogen bond with the adjacent Asn, which is highly conserved among many plant and fungal peroxidases. Our previous work [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., & Morishima, I. (1996) Biochemistry 35, 14251-14258] has revealed that the replacement of Asn70 in horseradish peroxidase C (HRP) by Val (N70V) and Asp (N70D) discourages the oxidation activity for guaiacol, and the elementary reaction rate constants for the mutants was decreased by 10-15-fold. In order to delineate the structure-function relationship of the His-Asn couple in peroxidase activity, heme environmental structures of the HRP mutant, N70D, were investigated by CD, 1H NMR, and IR spectroscopies as well as Fe2+/Fe3+ redox potential measurements. While N70D mutant exhibited quite similar CD spectra and redox potential to those of native enzyme, the paramagnetic NMR spectrum clearly showed that the hydrogen bond between the distal His and Asp70 is not formed in the mutant. The disappearance of the splitting in the 1H NMR signal of heme peripheral 8-methyl group observed in 50% H2O/50% D2O solution of N70D-CN suggests that the hydrogen bond between the distal His and heme-bound cyanide is also disrupted by the mutation, which was supported by the low C-N vibration frequency and large dissociation constant of the heme-bound cyanide in the mutant. Together with the results from various spectroscopies and redox potentials, we can conclude that the improper positioning of the distal His induced the cleavages of the hydrogen bonds around the distal His, resulting in the substantial decrease of the catalytic activity without large structural alterations of the enzyme. The His-Asn hydrogen bond in the distal site of peroxidases, therefore, is essential for the catalytic activity by controlling the precise location of the distal His.     

Implication of His68 in the substrate site of Bacillus subtilis adenylosuccinate lyase by mutagenesis and affinity labeling with 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate

Adenylosuccinate lyase of Bacillus subtilis is inactivated by 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate (2-BDB-TAMP) at pH 7.0. As the reagent concentration is increased, a maximum rate constant is approached, indicative of reversible enzyme-reagent complex formation (KR = 68 +/- 9 microM) prior to irreversible modification (kmax = 0.081 +/- 0.004 min-1). Complete inactivation occurs concomitant with about 1 mol of 2-BDB-[14C]TAMP incorporated/mol of enzyme subunit. Adenylosuccinate, or a combination of AMP and fumarate, decreases the inactivation rate and reduces incorporation of [14C] reagent, whereas either AMP or fumarate alone is much less effective. These observations suggest that 2-BDB-TAMP attacks the adenylosuccinate binding site. Proteolytic digestion of inactivated enzyme, followed by purification of the digest by HPLC, yields the radioactive peptide Ile62-Ala72, in which Arg67 and His68 are the most likely targets. Thus 2-BDB-TAMP reacts with adenylosuccinate lyase at a site distinct from the His141 attacked by 6-BDB-TAMP (Lee, Worby, Dixon, and Colman (1997) J. Biol. Chem. 272, 458-465). Site-directed mutagenesis was used to construct mutant enzymes with replacements for both Arg67 and His68, and either Arg67 or His68. The R67M mutant enzyme has almost the same specific activity as the wild-type enzyme under standard assay conditions, whereas the single mutant H68Q and double mutant R67M-H68Q enzymes exhibit specific activities that are decreased more than 100-fold. These results indicate that while Arg67 and His68 may both be in the region of the substrate site, only His68 is important for the catalytic activity of B. subtilis adenylosuccinate lyase. A role is proposed for His68 as a general acid-base catalyst.     

Reaction mechanism of fluoroacetate dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase (EC 3.8.1.3) catalyzes the dehalogenation of fluoroacetate and other haloacetates. The amino acid sequence of fluoroacetate dehalogenase from Moraxella sp. B is similar to that of haloalkane dehalogenase (EC 3.8.1.5) from Xanthobacter autotrophicus GJ10 in the regions around Asp-105 and His-272, which correspond to the active site nucleophile Asp-124 and the base catalyst His-289 of the haloalkane dehalogenase, respectively (Krooshof, G. H., Kwant, E. M., Damborsky, J., Koca, J., and Janssen, D. B. (1997) Biochemistry 36, 9571-9580). After multiple turnovers of the fluoroacetate dehalogenase reaction in H218O, the enzyme was digested with trypsin, and the molecular masses of the peptide fragments formed were measured by ion-spray mass spectrometry. Two 18O atoms were shown to be incorporated into the octapeptide, Phe-99-Arg-106. Tandem mass spectrometric analysis of this peptide revealed that Asp-105 was labeled with two 18O atoms. These results indicate that Asp-105 acts as a nucleophile to attack the alpha-carbon of the substrate, leading to the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule on the carbonyl carbon atom. A His-272 --> Asn mutant (H272N) showed no activity with either fluoroacetate or chloroacetate. However, ion-spray mass spectrometry revealed that the H272N mutant enzyme was covalently alkylated with the substrate. The reaction of the H272N mutant enzyme with [14C]chloroacetate also showed the incorporation of radioactivity into the enzyme. These results suggest that His-272 probably acts as a base catalyst for the hydrolysis of the covalent ester intermediate.     

Oxidation of corticosteroids to steroidal carboxylic acids by an enzyme preparation from hamster liver

Enzyme activity which catalyzes the oxidation of 11-deoxycorticosterone to 21-oic acids accompanies the "detritiating" enzyme (isomerase) of hamster liver recently isolated by Martin, K. O., et al. ((1977) Biochemistry 16 (preceding paper in this issue)). The metabolites isolated were 20alpha- and 20beta-hydroxy-3-oxo-pregn-4-en-21-oic acid and 3,20-dioxo-pregn-4-en-21-oic acid. When 21-hydroxy[4-14C, 21-3H]pregn-4-en-3,20-dione was the substrate, about half of the tritium was retained in position 20 of the hydroxy acids. The system which catalyzes the conversion of the ketol side chain of corticosteroids to acid metabolites appears to be a cluster of closely related enzymes. As a result of these studies, we believe that the hamster liver enzyme preparation provides a useful model system for studies on the biosynthesis of acid metabolites of the corticosteroids in man.     

H NMR probes for inter-segmental hydrogen bonds in myoglobins

NMR signals arising from the HisB5 N delta H and HisEF5 N epsilon H protons in sperm whale skeletal and horse heart myoglobins have been located for the first time in the downfield shifted portion of the spectra. The shifts and hydrogen exchange rates indicate that these His imidazole ring NH protons are involved in the inter-segmental hydrogen bonds of the protein in solution, as demonstrated by a crystallographic study [Takano, T. (1977) J. Mol. Biol. 220, 381-399]. The assigned His imidazole ring NH proton resonances can serve as new sensitive structural probes in the study of the local conformation of myoglobin. The applicability of the NMR spectral parameters in the study of the tertiary structure of apomyoglobin, the denaturation of the protein, and the protein stability of sperm whale and horse myoglobins is presented in some detail.     

Biosynthesis of pteridines. NMR studies on the reaction mechanisms of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, and sepiapterin reductase

GTP cyclohydrolase I catalyzes a ring expansion affording dihydroneopterin triphosphate from GTP. [1',2',3',4',5'-13C5, 2'-2H1]GTP was prepared enzymatically from [U-13C6]glucose for use as enzyme substrate. Multinuclear NMR experiments showed that the reaction catalyzed by GTP cyclohydrolase I involves the release of a proton from C-2' of GTP that is exchanged with the bulk solvent. Subsequently, a proton is reintroduced stereospecifically from the bulk solvent. This is in line with an Amadori rearrangement mechanism. The proton introduced from solvent occupies the pro-7R position in the enzyme product. The data also confirm that the reaction catalyzed by pyruvoyltetrahydropterin synthase results in the incorporation of solvent protons into positions C-6 and C-3' of the enzyme product. On the other hand, the reaction catalyzed by sepiapterin reductase does not involve any detectable incorporation of solvent protons into tetrahydrobiopterin.