Transition-state analysis of AMP deaminase

The transition state of the allosteric AMP deaminase from Saccharomyces cerevisiae has been characterized by 14C and 15N Vmax/Km heavy-atom kinetic isotope effects. The primary 6-14C isotope effect was measured with [6-14C]AMP, and the 6-15N primary isotope effect was measured by isotope ratio mass spectrometry using the natural abundance of 15N in AMP and by using 15N release from ATP as a slow substrate. Isotope effects for AMP as the substrate were measured in the presence and absence of ATP as an allosteric activator and GTP as an allosteric inhibitor. Kinetic isotope effects with [6-14C]AMP were 1.030 +/- 0.003, 1.038 +/- 0.004, and 1.042 +/- 0.003 in the absence of effectors and in the presence of ATP and GTP, respectively. Isotope effects for [6-15N]AMP averaged 1.010 +/- 0.002. Allosteric activation increased the 15N isotope effect to 1.016 +/- 0.003. A primary 15N kinetic isotope effect with ATP, which has a Vmax/Km 10(-6) that for AMP, was 1.013 +/- 0.001. The presence of D2O as solvent caused a marginally significant decrease in the [6-15N]AMP kinetic isotope effect from 1.011 +/- 0.001 to 1.007 +/- 0.002. Previous studies have established that the solvent D2O effect is inverse (0.34) for slow substrates with two or more protons transferred prior to transition state formation and remains inverse (0.79) with AMP as substrate [Merkler, D. J., & Schramm, V. L. (1993) Biochemistry 32, 5792-5799]. Bond vibrational analysis was used to identify transition states for AMP deaminase that are consistent with all kinetic isotope effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstruction of mitochondrial cytochrome P450-dependent steroid hydroxylases in artificial phospholipid membranes: studies of cytochrome P450scc topology by limited proteolysis

Highly purified cytochrome P450scc from bovine adrenal cortex mitochondria was inserted in artificial phospholipid membranes prepared from phosphatidylcholine to study the main principles of its membrane organization in the model system. Topology of the cytochrome P450scc polypeptide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of the products of proteolysis products of the membrane-bound heme protein. It is shown that limited proteolysis of cytochrome P450scc in proteoliposomes results in a significant decrease of Vmax for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted system in the presence of exogenously added adrenodoxin-reductase and adrenodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adrenodoxin is increased. Different models of membrane organization as well as functional specificity of cytochrome P450scc in artificial membranes are discussed.     

Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells

There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.     

Intragastric proteolysis after vagotomy in perforative duodenal ulcer

The examination of intragastric proteolysis in patients operated on the perforative duodenal ulcer has been performed in 80 patients in the period up to 16 years after the surgery. The selective proximal vagotomy was performed in 65 of the patients, the trunk vagotomy--in 15 of the patients. In 55 patients the vagotomy was combined with ulcerrraphy, in 25 patients--with draining operations. The 30-35% decrease of the intragastric pepsin proteolysis was detected in patients after the vagotomy. There was no any signs of restoration of the suppressed proteolytic activity in the time period up to 16 years after the surgery. The draining operations lead to suppression of pepsin proteolysis in long-term period after the surgery and to its substitution with digestion with the pancreatic proteinases.     

Effects of substitutions of the conserved histidine residues in human gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase possesses two histidine residues at positions 383 and 505 which are conserved in all mammalian and bacterial species. In order to elucidate the functions of these residues, we prepared mutants in which these residues were replaced by Ala. Kinetic analysis of the hydrolysis of L-gamma-glutamyl-p-nitroanilide indicated that substitution at His-383 decreased the Vmax value to 14% of that of the wild type, but had no effect on Vmax/K(m). In reactions involving glycylglycine as the acceptor substrate, the Vmax value of this mutant decreased to 38% with little alteration of Vmax/K(m) for L-gamma-glutamyl-p-nitroanilide as a gamma-glutamyl donor, but with a significant reduction of Vmax/K(m) for the acceptor. These results show that this substitution causes impairment of the step in which the free enzyme is regenerated from the gamma-glutamyl enzyme by water or an acceptor substrate. On the other hand, replacement of His-505 resulted in a decrease of the Vmax value for transpeptidation to about 10% of that of the wild type despite no substantial effect on the Vmax value for the hydrolysis reaction. However, this substitution did not affect Vmax/K(m) for the acceptor on transpeptidation. Thus, the formation of a non-productive enzyme-substrate complex with the acceptor substrate would decrease the Vmax value on transpeptidation. These results suggest that His-383 plays an important catalytic role in facilitating the degradation of the gamma-glutamyl-enzyme through hydrolysis or transfer of the gamma-glutamyl moiety to an acceptor. It was also shown that His-505 is important in the formation of a complex of the gamma-glutamyl enzyme with the acceptor substrate even though it plays no critical role in the catalysis. Although the pH-dependence profile and the van't Hoff plot for the ionic group responsible for enzyme activity were consistent with the requirement of a histidine residue, neither of the conserved histidines could be assigned as such an ionic group. This suggests that another histidine residue(s) might play an essential role in the enzyme function.     

Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase

Bovine serum amine oxidase (BSAO) catalyzes the oxidative deamination of primary amines, concomitant with the reduction of molecular oxygen to hydrogen peroxide via a ping-pong mechanism. A protocol has been developed for an analysis of chemical and kinetic mechanisms in the conversion of dioxygen to hydrogen peroxide. Steady-state kinetics show that two groups need to be deprotonated to facilitate the oxidative half-reaction. The pH dependence of Vmax/Km(O2) reveals pKa's of 6.2 +/- 0.3 and 7.0 +/- 0.2, respectively. A pKa of 7.2 +/- 0.1 has been obtained from a titration of anaerobically reduced BSAO using UV-vis spectrophotometry. The near identity of the pKa obtained from the reduced enzyme titration with the second pKa from steady-state kinetics suggests that this second pKa arises from the reduced cofactor. The assignment of pKa is supported by the observed pH dependence for formation of the cofactor semiquinone signal, detected by EPR spectroscopy under anaerobic conditions. To address the nature of rate-limiting steps in the oxidative half-reaction, the solvent isotope effect, viscosity effect, and O-18 isotope effect on Vmax/Km(O2) have been determined. The solvent isotope effect is indistinguishable from unity, ruling out a proton transfer as a rate-determining step. Use of glucose as a solvent viscosogen shows no viscosity effect, indicating that binding of oxygen is not in the rate-determining step. The O-18 kinetic isotope effect is independent of pH with an average value of 18(V/K) = 1.0097 +/- 0. 0010. This has been compared to calculated equilibrium O-18 isotope effects for various dioxygen intermediate species [Tian and Klinman (1993) J. Am. Chem. Soc. 115, 8891], leading to the conclusion that either the first electron transfer to dioxygen or the desorption of product peroxide from a Cu(II)-OOH complex could be the rate-limiting step. The distribution of steady-state enzyme species was, therefore, analyzed through a combination of stopped-flow experiments and analysis of DV and D(V/K) for benzylamine oxidation. We conclude that the major species accumulating in the steady state are the oxidized cofactor-substrate Schiff base complex and the reduced, aminoquinol form of cofactor. These data rule out a slow release of product hydroperoxide from the aminoquinone form of enzyme, leading to the conclusion that the first electron transfer from substrate-reduced cofactor to dioxygen is the rate-determining step in the oxidative half-reaction. This step is also estimated to be 40% rate-limiting in kcat. An important mechanistic conclusion from this study is that dioxygen binding is a separate step from the rate-limiting electron-transfer step to form superoxide. On the basis of a recently determined X-ray structure for the active form of a yeast amine oxidase from Hansenula polymorpha [Li et al. (1998) Structure 6, 293], a hydrophobic space has been identified near the O-2 position of reduced cofactor as the putative dioxygen binding site. Movement of superoxide from this site onto the Cu(II) at the active site may occur prior to further electron transfer from cofactor to superoxide.     

Induction of cytochrome P4503A by the antiglucocorticoid mifepristone and a novel hypocholesterolaemic drug

An acidic proteinase was purified from human kidney cortex. The enzyme showed a molecular mass of 31 kDa by SDS-PAGE, 36 kDa by gel filtration, and isoelectric points of 5.2 and 6.1. The optimum pH for hydrolysis of bovine hemoglobin was about 3.5. Reverse-phase HPLC analysis of the incubation mixture of the enzyme with human plasma showed the presence of an active peptide on rat uterus muscle with the same retention time as the methionyl-lysyl-bradykinin (MLBK) standard. The specific activities were 2.91 micrograms MLBK equivalent mg-1.min-1 at pH 3.5 and 2.15 micrograms MLBK equivalent mg-1.min-1 at pH 6.0. All the enzymatic activities of this human kidney proteinase were inhibited by pepstatin A. Intramolecularly quenched fluorogenic substrates with amino acid sequences of human kininogen were used to determine the cleavage points. On the N-terminal sequences (Abz-Leu-Met-Lys-Arg-Pro-Eddnp and Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp) the cleavage occurred at the Leu-Met linkage, and on the C-terminal sequences (Abz-Phe-Arg-Ser-Ser-Arg-Eddnp and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp) the cleavage occurred at the Arg-Ser linkage. Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp++ + was hydrolyzed by the renal acidic proteinase and yielded the peptide Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Abz-bradykinin). Kinectic parameters were determined using Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp (K(m) = 0.69 +/- 0.08 microM; Kcat = 0.052 +/- 0.0095 s-1; Kcat/K(m) = 0.075 +/- 0.005 microM-1.s-1) and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp (K(m) = 1.56 +/- 0.16 microM; Kcat = 0.0048 +/- 0.0001 s-1; Kcat/K(m) = 0.003 +/- 0.0003 microM-1.s-1). Human liver cathepsin D had no activity on C-terminal sequences and human pepsin hydrolyzed them at the Ser-Ser bond. The results suggest that the renal acid proteinase is distinct from human pepsin and human liver cathepsin D and releases MLBK from human kininogen.     

Effects of Plasmodium berghei infection on arteether metabolism and disposition

Arteether (AE) is primarily deethylated to dihydroqinghaosu (DQHS) in rats and humans. Conversion of AE to DQHS was impaired in microsomes from rats infected with Plasmodium berghei. The Km for AE was 175.1 +/- 49.1 and 124.4 +/- 115.1 mumol/l, and Vmax was 2.24 +/- 0.45 and 1.22 +/- 0.67 nmol AE formed/mg protein/min in control and infected microsomes (p < 0.05), respectively. Calculated intrinsic clearance (CLint = initial Vmax/Km) for AE was only 4% lower in infected microsomes. Apparent pharmacokinetic parameter estimates for AE using the isolated perfused rat liver demonstrated no differences (p > 0.05) in volume of distribution, clearance, and half-life between normal and infected animals. Malaria infection resulted in decreased biliary excretion of free AE and DQHS. The majority of AE is eliminated via biliary excretion of conjugated DQHS, which is approximately 500-fold higher than free DQHS and 75-fold higher than free AE on a molar basis.     

Preserved antioxidative defense of lipoproteins in renal failure and during hemodialysis

Contact to artificial surfaces during hemodialysis activates leukocytes, which then form oxidized arachidonic acid products and free radicals. This might promote the oxidative modification of low-density lipoproteins (LDL) that play a key role in the initiation of atherosclerosis. Thus, leukocyte activation could specifically contribute to the high mortality from atherosclerotic complications on long-term hemodialysis. Therefore monitored LDL and high-density lipoprotein (HDL) resistance to copper-stimulated oxidation in patients with end-stage renal disease on maintenance hemodialysis with cellulose acetate or polysulfone membranes (n = 12), in patients with chronic renal failure (n = 13) and in healthy controls (n = 12). Six of the dialysis patients were restudied during a single cuprophane dialysis. Circulating leukocytes were reversibly reduced early in hemodialysis with cellulose acetate (minimum, 83.6% +/- 7.4% of baseline values at 30 minutes after dialysis start), polysulfone (minimum, 80.4% +/- 10.5% at 15 minutes; P < 0.05) and cuprophane (minimum, 24.5% +/- 8.5% at 60 minutes; P < 0.0001). Despite the leukocyte activation, LDL oxidation lag time was not shortened in comparison with healthy controls and was even prolonged at the end of cellulose acetate (P < 0.05) and cuprophane (P < 0.05) dialysis. HDL oxidation lag time increased (12.6% +/- 0.9%; P < 0.0001) 15 to 60 minutes after start of hemodialysis and returned to predialysis values thereafter. In patients with chronic renal failure, the lag time of HDL oxidation was significantly prolonged (13.34 minutes +/- 0.9) compared with healthy controls (10.91 +/- 2.0 minutes; P < 0.01) as well as compared with the dialysis patients at baseline (9.9 minutes +/- 1.4; P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mucoadhesive polymers as platforms for peroral peptide delivery and absorption: synthesis and evaluation of different chitosan-EDTA conjugates

The purpose of the present study was to synthesize and evaluate mucoadhesive polymers, exhibiting a high capacity to bind bivalent cations which are essential co-factors for intestinal proteolytic enzymes. Under the formation of amide bonds, the complexing agent EDTA was covalently bound to the primary amino groups of chitosan. One gram of the resulting conjugate with the lowest amount of remaining free amino groups (0.1 +/- 0.03%; mean +/- SD, n = 3) based on free chitosan as 1.0 was capable of binding 1.4 +/- 0.1 mM calcium, 2.0 +/- 0.1 mM zinc and 1.9 +/- 0.03 mM cobalt (mean +/- SD, n = 3) under intestinal pH-conditions, respectively. Whereas proteolytic activity of the serine proteases trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1) and elastase (EC 3.4.21.36) could not be inhibited, proteolytic activity of the zinc proteases carboxypeptidase A (EC 3.4.17.1) and aminopeptidase N (EC 3.4.11.2) was strongly inhibited by the chitosan-EDTA conjugate. Moreover, it displays quick swelling properties in water and basic aqueous solutions. The adhesive force of the conjugate was even higher than of chitosan HCl. However, lowering the percentage of covalently attached EDTA on the polymer, leads to a significantly reduced adhesive force. According to these results, chitosan-EDTA conjugates exhibiting the lowest amount of remaining free amino groups, seem to be a useful tool in overcoming the enzymatic barrier for perorally administered therapeutic peptides.     

Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry

Microfabrication technology offers the opportunity to construct microfluidic modules which are designed to perform specific, dedicated functions. Here we report the construction of a microfabricated device for the generation and delivery by electroosmotic pumping of solvent gradients at nanoliter per minute flow rates. The device consists of three solvent reservoirs and channels which were etched in glass. Solvent gradients and solvent flows were generated by computer controlled differential electroosmotic pumping of aqueous and organic phase, respectively, from the solvent reservoirs. The device was integrated into an analytical system consisting of the solvent gradient delivery module, a reverse phase microcolumn and an electrospray ionization ion trap mass spectrometer (MS). The system was used for the analysis at high sensitivity of peptides and peptide mixtures generated by proteolytic digestion of proteins. We have measured an absolute limit of detection as low as 1 fmol and a concentration limit of detection at the 100 amol/microL level. The system was also successfully used for the identification of proteins separated by 1D and 2D gel electrophoresis. This was achieved by gradient frontal analysis of the peptide mixture generated by proteolysis of the respective proteins, and the automated generation and interpretation of collision-induced dissociation spectra.     

Identification of human cytochrome P450 isoforms involved in the 3-hydroxylation of quinine by human live microsomes and nine recombinant human cytochromes P450

Studies using human liver microsomes and nine recombinant human cytochrome P450 (CYP) isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) were performed to identify the CYP isoform(s) involved in the major metabolic pathway (3-hydroxylation) of quinine in humans. Eadie-Hofstee plots for the formation of 3-hydroxyquinine exhibited apparently monophasic behavior for all of the 10 different microsomal samples studies. There was interindividual variability in the kinetic parameters, as follows: 1.8-, 3.2- and 3.5-fold for K(m) Vmax and Vmax/K(m), respectively. The mean +/- S.D. values for K(m), Vmax and Vmax/K(m) were 106.1 +/- 19.3 microM, 1.33 +/- 0.48 nmol/mg protein/min and 12.8 +/- 5.1 microliters/mg protein/min, respectively. With 10 different human liver microsomes, the relationships between the 3-hydroxylation of quinine and the metabolic activities for substrates of the respective CYP isoforms were evaluated. The 3-hydroxylation of quinine showed an excellent correlation (r = 0.986, P < .001) with 6 beta-hydroxylation of testosterone, a marker substrate for CYP3A4. A significant correlation (r = 0.768, P < .01) between the quinine 3-hydroxylase and S-mephenytoin 4'-hydroxylase activities was also observed. However, no significant correlation existed between the 3-hydroxylation of quinine and the oxidative activities for substrates for CYP1A2 (phenacetin), 2C9 (diclofenac), 2D6 (desipramine) and 2E1 (chlorzoxazone). Ketoconazole and troleandomycin (inhibitors of CYP3A4) inhibited the 3-hydroxylation of quinine by human liver microsomes with respective mean IC50 values of 0.026 microM and 28.9 microM. Anti-CYP3A antibodies strongly inhibited quinine 3-hydroxylation, whereas weak inhibition was observed in the presence of S-mephenytoin or anti-CYP2C antibodies. Among the nine recombinant human CYP isoforms, CYP3A4 exhibited the highest catalytic activity with respect to the 3-hydroxylation of quinine, compared with the minor activity of CYP2C19 and little discernible or no effect of other CYP isoforms. Collectively, these data suggest that the 3-hydroxylation of quinine is mediated mainly by CYP3A4 and to a minor extent by CYP2C19. Other CYP isoforms used herein appear to be of negligible importance in this major pathway of quinine in humans.     

Serum concentrations of free and total insulin-like growth factor-I, IGF binding proteins -1 and -3 and IGFBP-3 protease activity in boys with normal or precocious puberty

OBJECTIVE: Circulating IGF-I and IGF binding protein-3 (IGFBP-3) levels both increase in puberty where growth velocity is high. The amount of free IGF-I is dependent on the IGF-I level and on the concentrations of the specific IGFBPs. Furthermore, IGFBP-3 proteolysis regulates the bioavailability of IGF-I. However, the concentration of free IGF-I and possible IGFBP-3 proteolytic activity in puberty has not previously been studied. SUBJECTS AND MEASUREMENTS: We investigated serum levels of easily dissociable IGF-I concentrations and ultrafiltrated free IGF-I levels by specific assays in 60 healthy boys and in 5 boys with precocious puberty before and during GnRH agonist treatment. In addition, total serum IGF-I, IGFBP-1 and IGFBP-3 levels as well as IGFBP-3 protease activity were determined. RESULTS: Free (dissociable and ultrafiltrated) IGF-I concentrations were significantly higher in pubertal boys than in prepubertal children and correlated significantly with the molar ratio between IGF-I and IGFBP-3 (r = 0.69, P < 0.0001 and r = 0.54, P = 0.0008, respectively) and inversely with IGFBP-1 (r = -0.47, P < 0.0001 and r = -0.43, P = 0.0003, respectively). Multiple regression analysis suggested that IGFBP-3 level, and not IGFBP-1, was the major determinant of the free IGF-I serum level in normal boys. Free IGF-I levels were elevated in boys with precocious puberty and decreased during GnRH treatment. IGFBP-3 proteolysis was constant throughout puberty (mean 20%). CONCLUSIONS: We conclude that easily dissociable and ultrafiltrated free IGF-I serum levels are increased in boys with normal and precocious puberty and suggest that the increased free IGF-I serum concentration in puberty primarily reflects changes in total concentrations of IGF-I and IGFBPs secondary to increased GH secretion, but that it is not influenced by changes in IGFBP-3 proteolysis.     

Substance P in the ventral pallidum: projection from the ventral striatum, and electrophysiological and behavioral consequences of pallidal substance P

1. Piroximone was administered orally (p.o.) and intravenously (i.v.) to male Beagle dog. In vitro, piroximone was incubated with dog liver microsomes. 2. Piroximone was metabolized in vivo to five metabolites (1-5) representing approximately 20% of the total administered dose. 3. The parent drug and its metabolites were totally eliminated in urine. 4. Reduced piroximone (piroximole), representing approximately 10% of the administered dose, was identified as the major metabolic product in vivo. 5. In vitro, piroximone was metabolized by dog liver microsomes to isonicotinic acid (1) and piroximole (4), with the same ratio as in vivo (1:4 = 0.2). The Michaelis-Menten parameters were determined for piroximole formation and were: Kmapp = 733 microM and Vmax app = 232 pmol/mg protein/min. 6. Comparison of the pharmacokinetics of piroximone and piroximole revealed that both compounds were very well absorbed (F = 93 +/- 7 and 89 +/- 8% respectively), slightly distributed (Vd app = 0.78 +/- 0.04 and 1.02 +/- 0.09 l/kg p.o., and 0.95 +/- 0.05 and 0.76 +/- 0.13 1/kg i.v. respectively) and excreted into urine to the same extent (UEx = 54.7 +/- 1.2 and 53.2 +/- 12.6% p.o., and 59.1 +/- 5.3 and 51.2 +/- 5.7% i.v. respectively), except that the clearance of piroximone was two-fold higher than that observed for piroximole (ClT = 7.77 +/- 1.35 and 4.12 +/- 0.44 ml/min/kg p.o., and 7.68 +/- 1.25 and 4.06 +/- 0.51 ml/min/kg i.v. respectively).     

Identification and characterization of human cytochrome P450 isoforms interacting with pimozide

Using human liver microsomes (HLMs) and recombinant human cytochrome P450 (CYP450) isoforms, we identified the major route of pimozide metabolism, the CYP450 isoforms involved, and documented the inhibitory effect of pimozide on CYP450 isoforms. Pimozide was predominantly N-dealkylated to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI). The formation rate of DHPBI showed biphasic kinetics in HLMs, which suggests the participation of at least two activities. These were characterized as high-affinity (K(m1) and Vmax1) and low-affinity (K(m2) and Vmax2) components. The ratio of Vmax1 (14 pmol/min/mg protein)/K(m1) (0.73 microM) was 5.2 times higher than the ratio of Vmax2 (244 pmol/min/mg protein)/K(m2) (34 microM). K(m2) was 91 times higher than K(m1). The formation rate of DHPBI from 25 microM pimozide in nine human livers correlated significantly with the catalytic activity of CYP3A (Spearman r = 0.79, P = .028), but not with other isoforms. Potent inhibition of DHPBI formation from 10 microM pimozide was observed with ketoconazole (88%), troleandomycin (79%), furafylline (48%) and a combination of furafylline and ketoconazole (96%). Recombinant human CYP3A4 catalyzed DHPBI formation from 10 microM pimozide at the highest rate (V = 2.2 +/- 0.89 pmol/min/pmol P450) followed by CYP1A2 (V = 0.23 +/- 0.08 pmol/min/pmol P450), but other isoforms tested did not. The K(m) values derived with recombinant CYP3A4 and CYP1A2 were 5.7 microM and 36.1 microM, respectively. Pimozide itself was a potent inhibitor of CYP2D6 in HLMs when preincubated for 15 min (Ki = 0.75 +/- 0.98 microM) and a moderate inhibitor of CYP3A (Ki = 76.7 +/- 34.5 microM), with no significant effect on other isoforms tested. Our results suggest that pimozide metabolism is catalyzed mainly by CYP3A, but CYP1A2 also contributes. Pimozide metabolism is likely to be subject to interindividual variability in CYP3A and CYP1A2 expression and to drug interactions involving these isoforms. Pimozide itself may inhibit the metabolism of drugs that are substrates of CYP2D6.     

Gliclazide hydroxylation by rat liver microsomes

1. The metabolism of gliclazide to hydroxygliclazide has been investigated in Sprague-Dawley rat liver microsomes. 2. The kinetics of hydroxygliclazide formation are consistent with Michaelis-Menten kinetics (mean (+/- SD, n = 3) apparent K(m) and Vmax = 256 +/- 27 microM and 1.85 +/- 0.10 nmol/ min/mg respectively). 3. Tolbutamide competitively inhibited hydroxygliclazide formation (Ki = 840 microM) and gliclazide competitively inhibited hydroxytolbutamide formation (Ki = 240 microM) with Ki similar to K(m). Therefore gliclazide and tolbutamide may be metabolized by the same enzyme in the rat. In nine livers the formation of hydroxygliclazide correlated with the formation of hydroxytolbutamide (rs = 0.82, p < 0.01). 4. Diclofenac (Ki = 64 microM), phenytoin (Ki = 38 microM), mephenytoin (Ki = 66 microM), glibenclamide (Ki = 14 microM) and glipizide (Ki = 189 microM) were fully competitive inhibitors of gliclazide hydroxylation. The rank order of Ki constants differed for gliclazide and tolbutamide suggesting that gliclazide and tolbutamide hydroxylases are not identical enzymes. 5. Quinine (Ki = 0.3 microM) and quinidine (Ki = 4.3 microM) were partially competitive inhibitors of hydroxygliclazide formation. Hydroxylation of gliclazide was related to the activity of CYP2D1 as assessed by dextrorphan production from dextromethorphan (rs = 0.83, p = 0.01). 6. In the rat gliclazide is metabolized to hydroxygliclazide by at least two cytochrome P450 isoforms, including tolbutamide hydroxylase and 2D1, which have similar affinities for gliclazide.     

Thiol ester hydrolysis catalyzed by glutathione S-transferase A1-1

rGSTA1-1 has been shown to catalyze the hydrolysis of the thiol ester glutathionyl ethacrynate (E-SG). In contrast, neither the retro-Michael addition with the substrate EA-SG, to yield GSH and ethacrynic acid (EA), nor the conjugation reaction between GSH and EA to yield the thiol ester E-SG was catalyzed to any measurable extent under similar conditions. The steady state kcat and KM for hydrolysis of E-SG by wild type rGSTA1-1 were 0.11 +/- 0.009 min-1 and 15.7 +/- 1.6 mM, respectively. The site-directed mutant, Y9F, in which the catalytic Tyr-9 is substituted with Phe, was completely inactive in this reaction. To uncover a mechanistic signature that would distinguish between direct hydrolysis and covalent catalysis involving acylation of Tyr-9, solvent isotope exchange and mass spectrometry experiments were performed. No 18O incorporation into the starting thiol ester was detected with initial velocity solvent isotope exchange experiments. However, covalent adducts corresponding to acylated protein also were not observed by electrospray ionization mass spectrometry, even with an assay that minimized the experimental dead time and which allowed for detection of N-acetyltyrosine acylated with EA in a chemical model system. The kon and koff rate constants for association and dissociation of E-SG were determined, by stopped flow fluorescence, to be 5 x 10(5) s-1 M-1 and 6.7 s-1, respectively. Together with the isotope partitioning results, these rate constants were used to construct partial free energy profiles for the GST-catalyzed hydrolysis of E-SG, assuming that Tyr-9 acts as a general acid-base catalyst. The "one-way flux" of the thiol esterase reaction results directly from the thermodynamic stability of the products after rate-limiting attack of the thiol ester by H2O or Tyr-9, and is sufficient to drive the hydrolysis to completion, in contrast to GST-catalyzed breakdown of other GSH conjugates.     

Probing the mechanism of inosine monophosphate dehydrogenase with kinetic isotope effects and NMR determination of the hydride transfer stereospecificity

The mechanism of human type II inosine monophosphate dehydrogenase has been probed by measurements of primary deuterium kinetic isotope effects, and by determination of the stereochemical course of the reaction. The deuterium isotope effects on Vmax from [2-deutero]-IMP are unity for reactions with a variety of monovalent cation activators (K+, NH4+, Na+, Rb+) of various efficacy. In each case normal effects on Vmax/K(m) in the range of 1.9 to 3.5 are observed for both IMP and NAD, and are larger for NAD. These results demonstrate that both substrates can dissociate from the E.M+.IMP.NAD complex, therefore the kinetic mechanism is not ordered as previous steady-state kinetic studies have suggested. Comparison of reaction rates in D2O and H2O show no 2H isotope effect on Vmax, and a < or = twofold decrease in Vmax/K(m); thus, a proton transfer from solvent is not rate-limiting in turnover. The NMR spectrum of the [4-deutero]NADH produced in the reaction of [2-deutero]-IMP and NAD shows that the hydrogen is transferred to the B, or pro-S, side of the nicotinamide ring. Presteady-state kinetic experiments reveal a burst of NADH formation in the first turnover, demonstrating that a late step in the mechanism is rate-limiting. The rate of the burst phase is reduced approximately twofold with [2-deutero]IMP as substrate, indicating that the hydride transfer step is kinetically significant early in the reaction.     

Beta-secondary and solvent deuterium kinetic isotope effects on catalysis by the Streptomyces R61 DD-peptidase: comparisons with a structurally similar class C beta-lactamase

Beta-secondary and solvent deuterium kinetic isotope effects have been determined for the steady-state kinetic parameters V/K and V for turnover of a series of acyclic substrates by the DD-peptidase of Streptomyces R61 and the class C beta-lactamase of Enterobacter cloacae P99. Although these enzymes are evolutionarily related and have very similar tertiary and active site structure, they are functionally very different-the former efficiently catalyzes the hydrolysis of beta-lactams but not acyclic peptides while vice versa applies to the latter. The measured kinetic isotope effects reveal both similarities and differences in the steady-state transition states for turnover of the various substrates by these enzymes. In most cases, inverse beta-secondary isotope effects were observed, reflecting typical acyl-transfer transition states. With one substrate, however, m-[[(phenylacetyl)glycyl]oxy]benzoic acid, isotope effects on V/K of very close to unity were obtained for both enzymes. These were interpreted in terms of acylation transition state conformations where the extent of beta-CH hyperconjugation was similar to that in the free substrate. Differences in deacylation transition states (V) between the two enzymes with this substrate were interpreted in terms of different acyl-enzyme conformations. Solvent deuterium kinetic isotope effects on V/K were uniformly small, some even inverse, for both enzymes and with all substrates tested. At face value, this suggests the counterintuitive conclusion that little proton transfer occurs in acylation transition states in all of these instances. Closer analysis, however, suggests that for ester and amide (and probably beta-lactam) substrates, this result probably arises from an increase in proton fractionation factors on substrate binding being offset by their decrease in the acylation transition state. The former event derives from proton rearrangement on substrate binding and the latter, presumably, from general acid/base catalysis. This result may be general to all beta-lactam-recognizing enzymes. The solvent isotope effects also suggest that, at least for the P99 beta-lactamase, the acylation transition state of a thioester substrate does not involve proton transfer. This can be interpreted in terms of the rate-determining breakdown of a tetrahedral intermediate where no protonation of the leaving thiolate is required. Deacylation transition states of both enzymes appear to involve significant proton transfer, presumably arising from general acid/base catalysis.     

Binding of chloromethyl ketone substrate analogues to crystalline papain

Papain (EC 3.4.22.2) is a proteolytic enzyme, the three-dimensional structure of which has been determined by x-ray diffraction at 2.8 A resolution (Drenth, J., Jansonius, J.N., Koekoek, R., Swen, H. M., and Wothers, B.G. (1968), Nature (London) 218, 929-932). The active site is a groove on the molecular surface in which the essential sulfhydryl group of cysteine-25 is situated next to the imidazole ring of histidine-159. The main object of this study was to determine by the difference-Fourier technique the binding mode for the substrate in the groove in order to explain the substrate specificity of the enzyme (P2 should have a hydrophobic side chain (Berger and Schechter, 1970) and to contribute to an elucidation of the catalytic mechanism. To this end, three chloromethyl ketone substrate analogues were reacted with the enzyme by covalent attachment to the sulfur atom of cysteine-25. The products crystallized isomorphously with the parent structure that is not the native, active enzyme but a mixture of oxidized papain (probably papain-SO2-) and papain with an extra cysteine attached to cysteine-25. Although this made the interpretation of the difference electron density maps less easy, it provided us with a clear picture of the way in which the acyl part of the substrate binds in the active site groove. The carbonyl oxygen of the P1 residue is near two potential hydrogen-bond donating groups, the backbone NH of cysteine-25 and the NH2 of glutamine-19. Valine residues 133 and 157 are responsible for the preference of papain in its substrate splitting. By removing the methylene group that covalently attaches the inhibitor molecules to the sulfur atom of cysteine-25 we obtained acceptable models for the acyl-enzyme structure and for the tetrahedral intermediate. The carbonyl oxygen of the P1 residue, carrying a formal negative charge in the tetrahedral intermediate, is stabilized by formation of two hydrogen bonds with the backbone NH of cysteine-25 and the NH2 group of glutamine-19. This situation resembles that suggested for the proteolytic serine enzymes (Henderson, R., Wright, C. S., Hess, G. P., and Blow, D. M. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 63-70; Robertus, J. D., Kraut, J., Alden, R. A., and Birktoft, J. J. (1972b), Biochemistry 11, 4293-4303). The nitrogen atom of the scissile peptide bond was found close to the imidazole ring of histidine-159, suggesting a role for this ring in protonating the N atom of the leaving group (Lowe, 1970). This proton transfer would be facilitated by a 30 degrees rotation of the ring around the C beta-Cgamma bond from an in-plane position with the sulfur atom to an in-plane position with the N atom. The possibility of this rotation is derived from a difference electron-density map for fully oxidizied papain vs. the parent protein.