Alternate substrate inhibitors of an alpha-chymotrypsin: enantioselective interaction of aryl-substituted enol lactones

Four enol lactones, bearing phenyl or 1-naphthyl substituents on the alpha or beta positions [3-phenyl-6-methylenetetrahydro-2-pyranone (alpha Ph6H, IIc), 3-(1-naphthyl)-6-methylenetetrahydro-2-pyranone (alpha Np6H, IId), 4-phenyl-6-methylenetetrahydro-2-pyranone (beta Ph6H, IIIc), and 4-(1-naphthyl)-6-methylenetetrahydro-2-pyranone (beta Np6H, IIId)], available as pure R and S enantiomers, have been studied as alternate substrate inhibitors of chymotrypsin. Kinetic constants for substrate binding (Ks) and acylation (ka) were determined by a competitive substrate assay, using succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide; the deacylation rate constant (kd) was determined by the proflavin displacement assay. All lactones undergo rapid acylation (ka varies from 17 to 170 min-1) that shows little enantioselectivity; there is, however, pronounced enantioselectivity in substrate binding for three of the lactones [Ks(R/S) = 40-110]. In each case it is the enantiomer with the S configuration that has the higher affinity. In all cases, deacylation rates are slow, and in two cases, acyl enzymes with half-lives of 4.0 and 12.5 h at pH 7.2, 25 degrees C, are obtained (for beta Ph6H and alpha Np6H, respectively). In these cases, high deacylation enantioselectivity is observed [kd(S/R) = 60-70], and the lactone more weakly bound as a substrate (R enantiomer) gives the more stable acyl enzyme. Two hypotheses, involving hindrance of the attack of water or an exchange of the ester and ketone carbonyl groups in the acyl enzyme, are advanced as possible explanations for the high stability of these acyl enzymes.     

Inhibition by bestatin of a mouse ascites tumor dipeptidase. Reversal by certain substrates

Bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl]-L-leucine, a known inhibitor of aminopeptidases, is shown to be a potent linear competitive inhibitor (KI,2.7 nM) of a dipeptidase purified from Ehrlich-Lettré hyperdiploid mouse ascites tumor cells. This inhibition can be classified as "slow binding" but not as "tight binding." Substrate protects the enzyme from bestatin inhibition when enzyme and inhibitor are in approximately equimolar concentrations. Addition of substrate (6 mM) partially (by about 20%) reverses dipeptidase inhibition by bestatin, but the time required for maximum recovery depends on the nature of the substrate. Substrates with lower Km (0.28-1.4 mM) values that exhibit substantial substrate inhibition require longer times (23-65 min) than those with higher Km values that show little substrate inhibition. Substrates with Km values higher than 1.5 mM do not reverse inhibition. The inhibition of the tumor dipeptidase by bestatin has been compared with inhibition by a variety of inhibitors of other Zn-metallo-proteolytic enzymes. These inhibitors were far less potent (KI, 0.063-10 mM), indicating a difference between the tumor dipeptidase and other enzymes of that class. Our results are discussed in terms of a postulated model of the bestatin molecule in the active site of the tumor dipeptidase, an enzyme which has not been studied by x-ray crystallographic means. The phenyl group of bestatin is placed in a hydrophobic pocket that is external but adjacent to the active site of the tumor dipeptidase. The shape of this pocket, as it appears from our results plus modeling, is such that only certain R groups of substrate can fit. The existence of such a pocket might explain the differential effect of substrates in the reversal of bestatin inhibition of the dipeptidase and also might explain substrate inhibition by misalignment of R groups into this pocket.     

Substrate specificity analysis and inhibitor design of homoisocitrate dehydrogenase

Homoisocitrate dehydrogenase is involved in the alpha-aminoadipate pathway of biosynthesis of l-lysine in fungi, yeast, some prokaryotic bacteria, and archaea. This enzyme catalyzes the oxidative decarboxylation of (2R,3S)-homoisocitrate into 2-oxoadipate using NAD(+) as a coenzyme. Substrate specificity of two homoisocitrate dehydrogenases derived from Deinococcus radiodurans and Saccharomyces cerevisiae was analyzed using a series of synthetic substrate analogs, which indicated a relatively broad substrate specificity of these enzymes. Based on the substrate specificity, 3-hydroxyalkylidene- and 3-carboxyalkylidenemalate derivatives were designed as a specific inhibitor for homoisocitrate dehydrogenase. The synthetic inhibitors showed a moderate competitive inhibitory activity and (R,Z)-3-carboxypropylidenemalate was the most inhibitory among the synthesized inhibitors. Therefore, homoisocitrate dehydrogenase appeared to recognize preferentially an extended conformation of homoisocitrate.     

Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli.

Crystal structures of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli have been determined to 2.0-A resolution in the absence of ligands, and to 2.5-A resolution with the feedback inhibitor AMP bound to the PRPP catalytic site. Glutamine PRPP amidotransferase (GPATase) employs separate catalytic domains to abstract nitrogen from the amide of glutamine and to transfer nitrogen to the acceptor substrate PRPP. The unliganded and AMP-bound structures, which are essentially identical, are interpreted as the inhibited form of the enzyme because the two active sites are disconnected and the PRPP active site is solvent exposed. The structures were compared with a previously reported 3.0-A structure of the homologous Bacillus subtilis enzyme (Smith JL et al., 1994, Science 264:1427-1433). The comparison indicates a pattern of conservation of peptide structures involved with catalysis and variability in enzyme regulatory functions. Control of glutaminase activity, communication between the active sites, and regulation by feedback inhibitors are addressed differently by E. coli and B. subtilis GPATases. The E. coli enzyme is a prototype for the metal-free GPATases, whereas the B. subtilis enzyme represents the metal-containing enzymes. The structure of the E. coli enzyme suggests that a common ancestor of the two enzyme subfamilies may have included an Fe-S cluster.     

Ability of Bacillus subtilis protoplasts to repair irradiated bacteriophage deoxyribonucleic acid via acquired and natural enzymatic systems.

A novel form of "enzyme therapy" was achieved by utilizing protoplasts of Bacillus subtilis. Photoreactivating enzyme of Escherichia coli was successfully inserted into the protoplasts of B. subtilis treated with polyethylene glycol. This enzyme was used to photoreactivate ultraviolet-damaged bacteriophage deoxyribonucleic acid (DNA). Furthermore, in polyethylene glycol-treated protoplasts, ultraviolet-irradiated transfecting bacteriophage DNA was shown to be a functional substrate for the host DNA excision repair system. Previous results (R. E. Yasbin, J. D. Fernwalt, and P. I. Fields, J. Bacteriol. 137:391-396, 1979) showed that ultraviolet-irradiated bacteriophage DNA could not be repaired via the excision repair system of competent cells. Therefore, the processing of bacteriophage DNA by protoplasts and by competent cells must be different. This sensitive protoplast assay can be used to identify and to isolate various types of DNA repair enzymes.     

Covalent modification of subtilisin Bacillus lentus cysteine mutants with enantiomerically pure chiral auxiliaries causes remarkable changes in activity

Methanethiosulfonate reagents may be used to introduce virtually unlimited structural modifications in enzymes via reaction with the thiol group of cysteine. The covalent coupling of enantiomerically pure (R) and (S) chiral auxiliary methanethiosulfonate ligands to cysteine mutants of subtilisin Bacillus lentus induces spectacular changes in catalytic activity between diastereomeric enzymes. Amidase and esterase kinetic assays using a low substrate approximation were used to establish kcat/KM values for the chemically modified mutants, and up to 3-fold differences in activity were found between diastereomeric enzymes. Changing the length of the carbon chain linking the phenyl or benzyl oxazolidinone ligand to the mutant N62C by a methylene unit reverses which diastereomeric enzyme is more active. Similarly, changing from a phenyl to benzyl oxazolidinone ligand at S166C reverses which diastereomeric enzyme is more active. Chiral modifications at S166C and L217C give CMMs having both high esterase kcat/KM's and high esterase to amidase ratios with large differences between diastereomeric enzymes.     

3-Isopropylmalate is the major endogenous substrate of the Saccharomyces cerevisiae trans-aconitate methyltransferase

The Saccharomyces cerevisiae Tmt1 gene product is the yeast homologue of the Escherichia coli enzyme that catalyzes the methyl esterification of trans-aconitate, a thermodynamically favored isomer of cis-aconitate and an inhibitor of the citric acid cycle. It has been proposed that methylation may attenuate trans-aconitate inhibition of aconitase and other enzymes of the cycle. Although trans-aconitate is a minor endogenous substrate of the Tmt1 enzyme in extracts of S. cerevisiae, the major endogenous substrate has yet to be identified. We show here that a trimethylsilylated derivative of the major methylated endogenous product of Tmt1 in yeast extracts has an identical gas chromatography retention time and an identical electron impact mass spectrum as one of the two possible monomethyl ester derivatives of (2R,3S)-3-isopropylmalate. (2R,3S)-3-Isopropylmalate is an intermediate of the leucine biosynthetic pathway that shares similar intermediates and reaction chemistry with the portion of the citric acid cycle from oxaloacetate to alpha-ketoglutarate via cis-aconitate. The Tmt1 methyltransferase recognizes (2R,3S)-3-isopropylmalate with similar kinetics as it does trans-aconitate, with respective K(m) values of 127 and 53 microM and V(max) values of 59 and 70 nmol min(-1) mg(-1) of protein in a Tmt1-overexpressed yeast extract. However, we found that isopropylfumarate, the direct homologue of trans-aconitate in the leucine biosynthetic pathway, was at best a very poor substrate for the Tmt1 yeast enzyme. Similarly, the direct homologue of 3-isopropylmalate in the citric acid cycle, isocitrate, is also a very poor substrate. This apparent change in specificity between the intermediates of these two pathways can be understood in terms of the binding of these substrates to the active site. These results suggest that the Tmt1 methyltransferase may work in two different pathways in two different ways: for detoxification in the citric acid cycle and for a possibly novel biosynthetic branch reaction of the leucine biosynthetic pathway.     

Benzylic alcohols as stereospecific substrates and inhibitors for aryl sulfotransferase

Aryl sulfotransferase IV catalyzes the 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent formation of sulfuric acid esters of benzylic alcohols. Since the benzylic carbon bearing the hydroxyl group can be asymmetric, the possibility of stereochemical control of substrate specificity of the sulfotransferase was investigated with benzylic alcohols. Benzylic alcohols of known stereochemistry were examined as potential substrates and inhibitors for the homogeneous enzyme purified from rat liver. For 1-phenylethanol, both the (+)-(R)- and (-)-(S)-enantiomers were substrates for the enzyme, and the kcat/Km value for the (-)-(S)-enantiomer was twice that of the (+)-(R)-enantiomer. The enzyme displayed an absolute stereospecificity with ephedrine and pseudoephedrine, and with 2-methyl-1-phenyl-1-propanol; that is, only (-)-(1R,2S)-ephedrine, (-)-(1R,2R)-pseudoephedrine, and (-)-(S)-2-methyl-1-phenyl-1-propanol were substrates for the sulfotransferase. In the case of 1,2,3,4-tetrahydro-1-naphthol, only the (-)-(R)-enantiomer was a substrate for the enzyme. Both (+)-(R)-2-methyl-1-phenyl-1-propanol and (+)-(S)-1,2,3,4-tetrahydro-1-naphthol were competitive inhibitors of the aryl sulfotransferase-catalyzed sulfation of 1-naphthalenemethanol. Thus, the configuration of the benzylic carbon bearing the hydroxyl group determined whether these benzylic alcohols were substrates or inhibitors of the rat hepatic aryl sulfotransferase IV. Furthermore, benzylic alcohols such as (+)-(S)-1,2,3,4-tetrahydro-1-naphthol represent a new class of inhibitors for the aryl sulfotransferase.     

Optically active 2-benzyl-3-methanesulfinylpropanoic acid: Synthesis and evaluation as inhibitors for carboxypeptidase A

All four possible stereomers of 2-benzyl-3-methanesulfinylpropanoic acid were synthesized and evaluated as inhibitors for carboxypeptidase A to find that the isomer having the (2S,4S)-configuration is most potent followed by isomers of (2R,4S)- and (2S,4R)-configurations. The stereochemical preferences shown by the isomers of the inhibitor in binding to the enzyme suggest that the sulfoxide oxygen in the inhibitor fails to ligate the active site zinc ion but may form a hydrogen bond with the guanidinium moiety of Arg-127 like the carbonyl oxygen of scissile peptide bond of oligopeptide substrate of the enzyme does. It may thus be inferred that a sulfoxide moiety may serve as an isosterer of a carboxamide moiety.     

Chemical synthesis, absolute configuration, and stereochemistry of formation of 10-hydroxywarfarin: a major oxidative metabolite of (+)-(R)-warfarin from hepatic microsomal preparations

The synthesis of a diastereomerically pure 10-hydroxywarfarin [4-hydroxy-3-(2-hydroxy-3-oxo-1-phenylbutyl)-2H-1 benzopyran-2-one] was accomplished in three steps from racemic warfarin. The relative configuration of the synthetic product was established by conversion to a cyclic derivative followed by NMR and X-ray diffraction analysis. Absolute stereochemistry was determined by enzymatic conversion of either of the pure enantiomers of warfarin to a 10-hydroxy metabolite of known relative configuration. Metabolic formation of 10-hydroxywarfarin was studied using hepatic microsomal preparations from female rats and man. The formation of 10-hydroxywarfarin catalyzed by hepatic microsomes from both dexamethasone-treated rats and man was highly stereoselective [(R)/(S): 3.4-9.0] for (R)-warfarin. In contrast, little stereoselectivity was observed in reactions catalyzed by untreated rat liver microsomes. The resultant stereochemistry at the site of oxidation was also found to be highly dependent on substrate stereochemistry. (R)-Warfarin gave (9R;10S)-10-hydroxywarfarin with only a trace of the (9R;10R) isomer irrespective of which enzyme preparation was used for catalysis, while (S)-warfarin gave (9S;10R)-10-hydroxywarfarin with only a trace of the (9S;10S) isomer, again irrespective of which enzyme preparation was used for catalysis.     

Biosynthesis of monoterpenes: stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to camphane and isocamphane monoterpenes

The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (-)-bornyl pyrophosphate and (-)-camphene, isomerization of the substrate to the (+)-(3S)-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia officinalis (sage) and Tanacetum vulgare (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3R)-[1Z-3H]linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)-camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3S)-[1Z-3H]-linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate yielded double-labeled (-)-bornyl pyrophosphate and (-)-camphene. Each product was then transformed to the corresponding (+)- or (-)-camphor without change in the 3H:14C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogen of the derived ketone. The finding that the 1Z-3H of the linalyl precursor was positioned at the endo-alpha-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at C1 of the geranyl substrate in these transformations, confirmed the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endo- conformer. These relative stereochemical elements, in combination with the observed enantiospecificities of the enzymes for the linalyl intermediates, allows definition of the overall absolute stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal camphane (bornane) and isocamphane monoterpenoids.     

Understanding substrate specificity in human and parasite phosphoribosyltransferases through calculation and experiment.

We present molecular dynamics (MD) simulations on two enzymes: a human hypoxanthine-guanine-phosphoribosyltransferase (HGPRTase) and its analogue in the protozoan parasite Tritrichomonas foetus. The parasite enzyme has an additional ability to process xanthine as a substrate, making it a hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) [Chin, M. S., and Wang, C. C. (1994) Mol. Biochem. Parasitol. 63 (2), 221-229 (1)]. X-ray crystal structures of both enzymes complexed to guanine monoribosyl phosphate (GMP) have been solved, and show only subtle differences in the two active sites [Eads et al. (1994) Cell 78 (2), 325-334 (2); Somoza et al. (1996) Biochemistry 35 (22), 7032-7040 (3)]. Most of the direct contacts with the base region of the substrate are made by the protein backbone, complicating the identification of residues significantly associated with xanthine recognition. Our calculations suggest that the broader specificity of the parasite enzyme is due to a significantly more flexible base-binding region, and rationalize the effect of two mutations, R155E and D163N, that alter substrate specificity [Munagala, N. R., and Wang, C. C. (1998) Biochemistry 37 (47), 16612-16619 (4)]. In addition, our simulations suggested a double mutant (D106E/D163N) that might rescue the D163N mutant. This double mutant was expressed and assayed, and its catalytic activity was confirmed. Our molecular dynamics trajectories were also used with a structure-based design program, Pictorial Representation Of Free Energy Changes (PROFEC), to suggest parasite-selective derivatives of GMP. Our calculations here successfully rationalize the parasite-selectivity of two novel inhibitors derived from the computer-aided design of Somoza et al. (5) and demonstrate the utility of PROFEC in the design of species-selective inhibitors.     

Perturbing the hydrophobic pocket of mandelate racemase to probe phenyl motion during catalysis

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Crystal structures of MR reveal that the phenyl group of all ground-state ligands is located within a hydrophobic cavity, remote from the site of proton abstraction. MR forms numerous electrostatic and H-bonding interactions with the alpha-OH and carboxyl groups of the substrate, suggesting that these polar groups may remain relatively fixed in position during catalysis while the phenyl group is free to move between two binding sites [i.e., the R-pocket and the S-pocket for binding the phenyl group of (R)-mandelate and (S)-mandelate, respectively]. We show that MR binds benzilate (K(i) = 0.67 +/- 0.12 mM) and (S)-cyclohexylphenylglycolate (K(i) = 0.50 +/- 0.03 mM) as competitive inhibitors with affinities similar to that which the enzyme exhibits for the substrate. Therefore, the active site can simultaneously accommodate two phenyl groups, consistent with the existence of an R-pocket and an S-pocket. Wild-type MR exhibits a slightly higher affinity for (S)-mandelate [i.e., K(m)(S)(-)(man) < K(m)(R)(-)(man)] but catalyzes the turnover of (R)-mandelate slightly more rapidly (i.e., k(cat)(R)(-->)(S) > k(cat)(S)(-->)(R)). Upon introduction of steric bulk into the S-pocket using site-directed mutagenesis (i.e., the F52W, Y54W, and F52W/Y54W mutants), this catalytic preference is reversed. Although the catalytic efficiency (k(cat)/K(m)) of all the mutants was reduced (11-280-fold), all mutants exhibited a higher affinity for (R)-mandelate than for (S)-mandelate, and higher turnover numbers with (S)-mandelate as the substrate, relative to those with (R)-mandelate. (R)- and (S)-2-hydroxybutyrate are expected to be less sensitive to the additional steric bulk in the S-pocket. Unlike those for mandelate, the relative binding affinities for these substrate analogues are not reversed. These results are consistent with steric obstruction in the S-pocket and support the hypothesis that the phenyl group of the substrate may move between an R-pocket and an S-pocket during racemization. These conclusions were also supported by modeling of the binary complexes of the wild-type and F52W/Y54W enzymes with the substrate analogues (R)- and (S)-atrolactate, and of wild-type MR with bound benzilate using molecular dynamics simulations.     

Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.     

Purification and characterization of two polymorphic variants of short chain acyl-CoA dehydrogenase reveal reduction of catalytic activity and stability of the Gly185Ser enzyme

Short chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric flavoenzyme that catalyzes the first intramitochondrial step in the beta-oxidation of fatty acids. Two polymorphisms in the coding region of the SCAD gene, 511C>T (R147W) and 625G>A (G185S), have been shown to be associated with an increased level of ethylmalonic acid excretion in urine, a clinical characteristic of SCAD deficiency. To characterize the biochemical consequences of these variations, in vitro site-directed mutagenesis and prokaryotic expression were used to produce the corresponding SCAD variant proteins. Both variant proteins were unstable when produced in Escherichia coli, but could be rescued and subsequently purified by coexpressing them with the bacterial chaperonin GroEL/ES. The k(cat)/K(m) values of the green wild-type, R147W, and G185S SCAD enzymes coexpressed with GroEL/ES were 33, 30, and 10 microM(-)(1) s(-)(1), respectively. There were minimal differences in the kinetic parameters measured for the green, degreened, and wild-type enzymes coexpressed with GroEL/ES, and the R147W variant when butyryl-CoA was used as a substrate. The catalytic efficiency of the G185S variant enzyme, however, was reduced compared to that of the wild-type enzyme. The thermal and guanidine HCl stability of the purified enzymes as determined by fluorescence, far-UV CD spectroscopy, and incubation-induced rest activity showed the following order of relative stability: wild-type enzyme > R147W > G185S. Near-UV CD spectroscopy indicated that these impairments are caused by decreased flexibility in the tertiary conformation of the two mutant enzymes. The common SCAD polymorphisms may lead to clinically relevant alterations in enzyme function.     

Urethanyl-3-amidinophenylalanine derivatives as inhibitors of factor Xa. X-ray crystal structure of a trypsin/inhibitor complex and modeling studies

Hydrophobic urethanyl derivatives of 3-amidinophenylalanine methyl ester were found to be relatively potent and selective factor Xa inhibitors. These compounds consist of the arginine-mimetic 3-benzamidino group as P1 residue and of hydrophobic residues as potential interaction partners for the S3/S4 aryl binding site of the enzyme. Attempts to possibly identify their binding mode to factor Xa via the X-ray crystal structure of a trypsin/inhibitor complex and analogy modeling on the crystal structure of factor Xa failed. However, synthesis of enantiomerically pure (R)- and (S)-derivatives, combined with modeling experiments, led to an hypothetical non-substrate like binding mode, which was fully confirmed by the remarkably enhanced inhibitory potency of derivatives in which the methyl ester was replaced by arylamides for interactions with the S3/S4 enzyme binding subsites. With adamantyloxycarbonyl-(R)-3-amidinophenylalanine-phenethylamide+ ++ a nanomolar inhibiton was obtained, thus indicating this new class of factor Xa inhibitors as a highly promising lead structure.     

Purification and characterization of (2S)-flavanone 3-hydroxylase from Petunia hybrida

(2S)-Flavanone 3-hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)-naringenin to (2R,3R)-dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2-oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200-fold by a six-step procedure with low recovery. The Mr of the enzyme was estimated by gel filtration to be about 74,000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2-oxoglutarate, Fe2+ and ascorbate. With 2-oxo[1-14C]glutarate in the enzyme assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)-(2R,3R)-dihydrokaempferol. (2S)-Naringenin, but not the (2R)-enantiomer is a substrate of the hydroxylase. (2S)-Eriodictyol is converted to (2R,3R)-dihydroquercetin. In contrast, 5,7,3',4',5'-pentahydroxy-flavanone is not a substrate. Apparent Michaelis constants for (2S)-naringenin and 2-oxoglutarate were determined to be respectively 5.6 mumol X l-1 and 20 mumol X l-1 at pH 8.5. The Km for (2S)-eriodictyol is 12 mumol X l-1 at pH 8.0. Pyridine 2,4-dicarboxylate and 2,5-dicarboxylate are strong competitive inhibitors with respect to 2-oxoglutarate with Ki values of 1.2 mumol X l-1 and 40 mumol X l-1, respectively.     

Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide.

A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-D-arginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (D-BAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. cereus T was inhibited by p-chloromercuribenzene sulfonic acid. The D-BAPAases of B. subtilis and B. cereus T had a Michaelis constant for D-BAPA of 2.9 x 10(-5) M and 1.4 x 10(-4) M, respectively. D-BAPAase is an intracellular enzyme localized in the protoplast (80 to 90% in soluble form in the cytoplasm). The ability to cleave D-BAPA is suggested as an additional chemotaxonomic characteristic of sporeforming bacteria of the genera Bacillus and Clostridium.     

Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phospholipase A2 with micellar lipid/water interfaces of competitive inhibitors.

In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257 and references therein) we have demonstrated that synthetic (R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A2 (PLA2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA2 and of an inactive PLA2 in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the (R)-configuration in its active site, whereas the (S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than those of the (S)-isomer. The modified PLA2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitory phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the large difference in overall dissociation constants of the complexes between enzyme and micelles of (R)-and (S)-inhibitors. With the use of PLA2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.     

Interconversion of E and S isoenzymes of horse liver alcohol dehydrogenase. Several residues contribute indirectly to catalysis.

The E and S isoenzymes of horse liver alcohol dehydrogenase differ by 10 amino acid residues, but only the S isoenzyme is active on 3 beta-hydroxysteroids. This functional difference was correlated to the differences in structures of the isoenzymes by characterizing a series of chimeric enzymes, which could represent intermediates in the evolution of catalytic activity. Deletion of Asp-115 from the E isoenzyme created the E/D115 delta enzyme that is active on steroids. The deletion alters the substrate binding pocket by moving Leu-116, which sterically hinders binding of steroids in the E isoenzyme. A chimeric enzyme (ESE) that has four changes in or near the substrate binding pocket (T94I/R101S/F110L/D115 delta) was 15-30-fold more catalytically efficient (V/Km) on uncharged steroids than was the E/D115 delta enzyme. Molecular modeling suggests that the substitutions at residues 94 and 110 indirectly affect the activity on steroids. ESE enzyme was 6-fold more active than the S isoenzyme on neutral steroids, due to substitutions not in the substrate binding pocket. The K366E and the Q17E/A43T/A59T substitutions in the S isoenzyme gave 2-fold increases in V/Km on steroids, which together can account for the changes observed with the ESE enzyme. The enzymes that are active on steroids did not bind 2,2,2-trifluoroethanol as tightly and were catalytically less efficient than the E isoenzyme with small alcohols. However, these enzymes were two to three and four to five orders of magnitude more efficient with 1-hexanol and 5 beta-androstane-3 beta,17 beta-diol, respectively, than with ethanol. These results demonstrate that several residues not directly participating in substrate binding or chemical catalysis contribute to catalytic efficiency.