The enzymatic formation of novel bile acid primary amides

Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O(2)-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The alpha-amidated peptides and the long-chain acylamides are hormones in humans and other mammals. Bile acid glycine conjugates are also substrates for PAM leading to the formation of bile acid amides. The (V(MAX)/K(m))(app) values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (V(MAX)/K(m))(app) value, 3.1 +/- 0.12 x 10(5) M(-1) s(-1) for 3-sulfolithocholylglycine, is 6.7-fold higher than that for d-Tyr-Val-Gly, a representative peptide substrate. The time course for O(2) consumption and glyoxylate production indicates that bile acid glycine conjugate amidation is a two-step reaction. The bile acid glycine conjugate is first converted to an N-bile acyl-alpha-hydroxyglycine intermediate which is ultimately dealkylated to the bile acid amide and glyoxylate. The enzymatically produced bile acid amides and the carbinolamide intermediates were characterized by mass spectrometry and two-dimensional (1)H-(13)C heteronuclear multiple quantum coherence NMR.     

Glutathione,S-substituted glutathiones,and leukotriene C4 as substrates for peptidylglycine alpha-amidating monooxygenase

The C-terminal alpha-amide moiety of most peptide hormones arises by the posttranslational cleavage of a glycine-extended precursor in a reaction catalyzed by bifunctional peptidylglycine alpha-amidating monooxygenase (PAM). Glutathione and the S-alkylated glutathiones have a C-terminal glycine and are, thus, potential substrates for PAM. The addition of PAM to glutathione, a series of S-alkylated glutathiones, and leukotriene C(4) results in the consumption of O(2) and the production of the corresponding amidated peptide and glyoxylate. This reaction proceeds in two steps with the intermediate formation of a C-terminal alpha-hydroxyglycine-extended peptide. Amidated glutathione (gammaGlu-Cys-amide) is a relatively poor substrate for glutathione S-transferase with a V/K value that is 1.3% of that for glutathione. Peptide substrates containing a penultimate hydrophobic or sulfur-containing amino acid exhibit the highest (V/K)(app) values for PAM-catalyzed amidation. The S-alkylated glutathiones incorporate both features in the penultimate position with S-decylglutathione having the highest (V/K)(app) of the substrates described in this report.     

Thiorphan, tiopronin, and related analogs as substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase (PAM)

Peptidyglycine alpha-amidating monooxygenase is a copper- and zinc-dependent, bifunctional enzyme that catalyzes the cleavage of glycine-extended peptides or N-acylglycines to the corresponding amides and glyoxylate. This reaction is a key step in the biosynthesis of bioactive alpha-amidated peptides and, perhaps, the primary fatty acids amides also. Two clinically useful N-acylglycines are thiorphan and tiopronin, each with a thiol moiety attached to the acyl group. We report here that thiorphan and tiopronin are substrates for PAM, exhibiting relatively low K(M,app) and V(MAX,app) values. The low V(MAX,app) values result, most likely, from a decrease in active PAM.2Cu(II) as the enzyme competes ineffectively with thiorphan and tiopronin for free copper.     

18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine alpha-amidating enzyme is a monooxygenase.

The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.     

Recombinant type A rat 75-kDa alpha-amidating enzyme catalyzes the conversion of glycine-extended peptides to peptide amides via an alpha-hydroxyglycine intermediate

The amidation of C-terminal glycine-extended peptides has been analyzed by the use of a truncated type A peptidylglycine alpha-amidating enzyme (alpha-AE) encoded by cDNA prepared with RNA from rat medullary thyroid carcinoma (MTC) cells. Mouse C127 cells transfected with the rat MTC cDNA encoding the truncated type A alpha-AE secrete the expected 75-kDa enzyme into the culture medium. Medium conditioned with the transfected C127 cells converts both dansyl-Tyr-Val-Gly and dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 at levels which are approximately 1000 times higher than the levels found in medium conditioned with untransfected C127 cells. This result indicates that rat type A alpha-AE alone catalyzes a two-step reaction involving an initial hydroxylation of peptidyl-Gly followed by conversion of the peptidyl-alpha-hydroxyglycine intermediate to the amidated product. The involvement of a separate, second enzyme to convert peptidyl-alpha-hydroxyglycine to peptidyl-NH2 is not necessary in this system. The initial hydroxylation step is rate-determining at infinite substrate concentration and requires a reducing equivalent, molecular oxygen, and copper.     

N-acylglycines: gas chromatographic mass spectrometric identification and determination in urine by selected ion monitoring

Eleven biologically interesting N-acylglycines have been synthesized and the gas chromatographic and mass spectrometric properties of their trimethylsilyl derivatives studied, A sharp and reproducible gas chromatographic peak could be obtained for each N-acylglycine as the N, O-bis-(trimethylsilyl)-N-acylglycine. By the use of these derivatives a sensitive and specific selected ion monitoring method for the determination of N-acylglycines in human urine has been developed.     

Reaction of serine-glyoxylate aminotransferase with the alternative substrate ketomalonate indicates rate-limiting protonation of a quinonoid intermediate

Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the interconversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The primary deuterium isotope effect using L-serine 2-D is one on (V/K)serine and V in the steady state. Pre-steady-state experiments also indicate that there is no primary deuterium isotope effect with L-serine 2-D. The results suggest there is no rate limitation by abstraction of the alpha proton of L-serine in the SGAT reaction. In the steady-state a solvent deuterium isotope effect of about 2 was measured on (V/K)L-serine and (V/K)ketomalonate and about 5.5 on V. Similar solvent isotope effects were observed in the pre-steady-state for the natural substrates and the alternative substrate ketomalonate. In the pre-steady-state, no reaction intermediates typical of PLP enzymes were observed with the substrates L-serine, glyoxylate, and hydroxypyruvate. The data suggest that breakdown and formation of the ketimine intermediate is the primary rate-limiting step with the natural substrates. In contrast, using the alternative substrate ketomalonate, pre-steady-state experiments display the transient formation of a 490 nm absorbing species typical of a quinonoid intermediate. The solvent isotope effect results also suggest that with ketomalonate as substrate protonation at C(alpha) is the slowest step in the SGAT reaction. This is the first report of a rate-limiting protonation of a quinonoid at C(alpha) of the external Schiff base in an aminotransferase reaction.     

Characterization of a bifunctional peptidylglycine alpha-amidating enzyme expressed in Chinese hamster ovary cells.

Peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the conversion of glycine-extended prohormones to their biologically active alpha-amidated forms. We have derived a clonal Chinese hamster ovary cell line that secretes significant quantities of active alpha-AE. Enzyme production was increased by selection for methotrexate-resistant cells expressing a dicistronic message. Amplification of the alpha-AE gene was monitored by Southern blot analysis, enzyme activity, and immunoreactive protein throughout the selection process. The soluble enzyme is bifunctional as determined by the ability to convert either the glycine-extended substrate, dansyl-Tyr--Val--Gly, or the intermediate, dansyl-Tyr--Val--alpha-hydroxyglycine, to the dansyl-Tyr--Val--NH2 product. The recombinant alpha-AE was purified by a simple two-step chromatographic process. The purified enzyme is partially glycosylated and the glycosylated and nonglycosylated forms of the enzyme were separated on a Con A-Sepharose column. The kinetic constants for dansyl-Tyr--Val--Gly, dansyl-Tyr--Val--alpha-hydroxyglycine, ascorbate, and catechol were the same for both forms of alpha-AE. In addition, mimosine is competitive vs ascorbate with K(is) = 3.5 microM for the nonglycosylated alpha-AE and K(is) = 4.2 microM for the glycosylated alpha-AE. Therefore, the presence or absence of asparagine-linked oligosaccharide does not affect the catalytic efficiency of the enzyme. Overexpression of the recombinant enzyme in CHO cells greatly enhances expression of the endogenous gene, implicating a feedback mechanism on the alpha-AE gene.     

Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test

Although aldolase-catalyzed condensations proceed by stepwise mechanisms via the intermediacy of nucleophilic enol(ate)s or enamines, the mechanisms of those enzymes that catalyze Claisen-type condensations are unclear. The reaction pathway followed by an enzyme from this second group, malate synthase, has been studied by the double-isotope fractionation method to determine whether the reaction is stepwise or concerted. In agreement with earlier work, a deuterium kinetic isotope effect D(V/K) of 1.3 +/- 0.1 has been found when [2H3]acetyl-CoA is the substrate. The 13C isotope effect at the aldehydic carbon of glyoxylate has also been measured. For this determination, the malate product (containing the carbon of interest at C-2) was quantitatively transformed into a new sample of malate having the carbon of interest at C-4. This material was decarboxylated by malic enzyme to produce the appropriate CO2 for isotope ratio mass spectrometric analysis. The 13C isotope effect with [1H3]acetyl-CoA [that is, 13(V/K)H] is 1.0037 +/- 0.0004. By use of the known values of the intermolecular and intramolecular deuterium effects and of 13(V/K)H, the value of the 13C isotope effect when deuteriated [2H3]acetyl-CoA is the substrate [that is, 13(V/K)D] can be predicted for three possible mechanisms. If 13(V/K)H is a kinetic isotope effect and the reaction is concerted, the value of the 13C effect on deuteriation of acetyl-CoA will rise to 1.011; if 13(V/K)H is a kinetic isotope effect and the reaction is stepwise, the value of the 13C effect will fall to 1.0025; and if the 13C effect is an equilibrium isotope effect deriving from glyoxylate dehydration, the reaction is necessarily stepwise, and the value of 13(V/K)D will be 1.0037, unchanged from that of 13(V/K)H. Experimentally, the value of 13(V/K)D is 1.0037 +/- 0.0007, which requires that malate synthase follow a stepwise path. It is therefore clear that the two salient characteristics of enzymes that catalyze Claisen-like condensations, namely, the absence of enzyme-catalyzed proton exchange with solvent and the inversion of the configuration at the nucleophilic center, which had been suggestive of a concerted pathway, are not mechanistically diagnostic.     

Kinetic and chemical mechanisms of the fabG-encoded Streptococcus pneumoniae beta-ketoacyl-ACP reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.     

Ubiquitin and ubiquitin-derived peptides as substrates for peptidylglycine alpha-amidating monooxygenase

Ubiquitin (Ub) and the ubiquitin-like proteins (UBLs) mediate an array of cellular functions. These proteins contain a C-terminal glycine residue that is key to their function. Oxidative conversion of C-terminal glycine-extended prohormones to the corresponding alpha-amidated peptide is one step in the biosynthesis of bioactive peptide hormones. The enzyme catalyzing this reaction is peptidylglycine alpha-amidating monooxygenase (PAM). We report herein that Ub is a PAM substrate with a (V/K)(amidation) that is similar to other known peptide substrates. This work is significant because PAM and the UBLs co-localize to the hypothalamus and the adrenal medulla and are both over-expressed in glioblastomas.     

Native chemical ligation derived method for recombinant peptide/protein C-terminal amidation

C-terminal amidation is often a requisite structural feature for peptide hormone bio-activity. We report a chemical amidation method that converts peptide/protein thioesters into their corresponding C-terminal amides. The peptide/protein thioester is treated with 1-(2,4-dimethoxyphenyl)-2-mercaptoethyl auxiliary (1b) in a native chemical ligation (NCL) reaction to form an intermediate, which upon removal of the auxiliary with TFA, yields the peptide/protein amide. We have demonstrated the general utility of the approach by successfully converting several synthetic peptide thioesters to peptide amides with high conversion rates. Preliminary results of converting a recombinant peptide thioester to its amide form are also reported.     

Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes

The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD(+) in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C(4)-proS position of the nicotinamide ring are small [(D)(V/K)(NADH) = 1.12 +/- 0.15, (D)V(app) = 1.05 +/- 0.07] when D,L-lipoamide is the oxidant but large and equivalent [(D)(V/K)(NADH) = (D)V = 2.95 +/- 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with (D)(V/K)(APADH) = 0.73 +/- 0.03, (D)(V/K)(Lip(S))2 = 0.77 +/- 0.03, and (D)V(app) = 0.77 +/- 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with (D)(V/K)(APADH) = 0.75 +/- 0.06, (D)(V/K)(DTP) = 0.71 +/- 0.02, and (D)V(app) = 0.56 +/- 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys(46) and Cys(41), limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.     

Crotonase-catalyzed beta-elimination is concerted: a double isotope effect study

Determining the sequence of bond cleavages, and consequently the nature of intermediates, in enzyme-catalyzed reactions is a major goal of mechanistic enzymology. When significant primary isotope effects on V/K are observed for two different bond cleavages, both bonds may be broken in the same transition state or they can reflect two different transition states that are of nearly identical energy and consequently both are partially rate limiting. For the crotonase-catalyzed dehydration of 3-hydroxybutyrylpantetheine, the primary D(V/K) and 18(V/K) are 1.60 and 1.053 [Bahnson, B. J., & Anderson, V. E. (1989) Biochemistry 28, 4173-4181], respectively. In this case, double isotope effects can discriminate between the two possibilities [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114; Belasco, J. G., Albery, W. J., & Knowles, J. R. (1983) J. Am. Chem. Soc. 105, 2475-2477]. The ratio of the alpha-secondary D(V/K) for the hydration of crotonylpantetheine catalyzed by crotonase in H2O and D2O has been determined to be 1.003 +/- 0.006. The invariance of the alpha-secondary effect where the chemical reaction is completely rate determining requires that both bond cleavages be concerted or that the substitution of 2H at the primary position not significantly alter the partitioning of a hypothetical carbanion. The observation of a solvent discrimination isotope effect determined from the relative incorporation of 2H from 50% D2O of 1.60 +/- 0.03, identical with the primary D(V/K), and the determination that the rate of exchange of the abstracted proton with solvent proceeds at less than 3% of the overall reaction rate also fail to provide evidence for a carbanion intermediate and are consistent with a concerted reaction. Identical primary D(V/K)s determined in H2O and D2O indicate that there is not a significant solvent isotope effect on C-O bond cleavage. The isotope ratios determined in these studies were performed by negative ion chemical ionization whole molecule mass spectrometry of the pentafluorobenzyl esters, a new method whose validity is established by comparison with previously determined kinetic and equilibrium isotope effects.     

Structural change of threonine 89 upon photoisomerization in bacteriorhodopsin as revealed by polarized FTIR spectroscopy.

The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O-H and N-H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899-7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm(-1) in bacteriorhodopsin and 2466 cm(-1) in the K intermediate, in D2O exhibited 18(O)-induced isotope shift. The O-H stretching vibrations of the threonine side chain correspond to 3378 cm(-1) in bacteriorhodopsin and to 3317 cm(-1) in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O-H stretch frequency of neat secondary alcohol is 3340-3355 cm(-1). The O-H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O-H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S-H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828-5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.     

Effects of vanadate on the oxidation of NADH by xanthine oxidase

Vanadate (V(V)) stimulates the oxidation of NADH by xanthine oxidase and superoxide dismutase eliminates the effect of V(V). Paraquat stimulates both the oxidation of NADH by xanthine oxidase and the V(V) enhancement of that oxidation. Xanthine, which is a better substrate for xanthine oxidase than is NADH, causes a V(V)-dependent co-oxidation of NADH which is transient and eliminated by SOD. Urate inhibits the V(V)-stimulated oxidation of NADH by xanthine oxidase or by Rose Bengal plus light. Measurement of rates of both O2- production and V(V)-stimulated NADH oxidation showed that many molecules of NADH were oxidized per O2-. These chain lengths were an inverse function of overall reaction rate. Minimum chain lengths, calculated on the basis of 100% univalent reduction of O2 to O2-, were smaller than measured average chain lengths by a factor of five. All of these results are in accord with the view that V(V) does not directly affect the activity of the enzyme, but rather catalyzes the free radical chain oxidation of NADH by O2-. It was further shown that phosphate was not involved and that the active form of V(V) was orthovanadate, rather than decavanadate.     

Structural requirements for expression of factor Va activity

Thrombin activated factor Va (factor VIIa, residues 1-709 and 1546-2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of approximately 0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1-696) and a Mr 80,000 light chain (amino acid residues 1509/1514-2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses approximately 60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by alpha-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app approximately 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.     

Chemical mechanism of the serine acetyltransferase from Haemophilus influenzae

The pH dependence of kinetic parameters was determined in both reaction directions to obtain information about the acid-base chemical mechanism of serine acetyltransferase from Haemophilus influenzae (HiSAT). The maximum rates in both reaction directions, as well as the V/K(serine) and V/K(OAS), decrease at low pH, exhibiting a pK of approximately 7 for a single enzyme residue that must be unprotonated for optimum activity. The pH-independent values of V(1)/E(t), V(1)/K(serine)E(t), V/K(AcCoA)E(t), V(2)/E(t), V(2)/K(OAS)E(t), and V/K(CoA)E(t) are 3300 +/- 180 s(-1), (9.6 +/- 0.4) x 10(5) M(-1) s(-1), 3.3 x 10(6) M(-1) s(-1), 420 +/- 50 s(-1), (2.1 +/- 0.5) x 10(4) M(-1) s(-1), and (4.2 +/- 0.7) x 10(5) M(-1) s(-1), respectively. The K(i) values for the competitive inhibitors glycine and l-cysteine are pH-independent. The solvent deuterium kinetic isotope effects on V and V/K in the direction of serine acetylation are 1.9 +/- 0.2 and 2.5 +/- 0.4, respectively, and the proton inventories are linear for both parameters. Data are consistent with a single proton in flight in the rate-limiting transition state. A general base catalytic mechanism is proposed for the serine acetyltransferase. Once acetyl-CoA and l-serine are bound, an enzymic general base accepts a proton from the l-serine side chain hydroxyl as it undergoes a nucleophilic attack on the carbonyl of acetyl-CoA. The same enzyme residue then functions as a general acid, donating a proton to the sulfur atom of CoASH as the tetrahedral intermediate collapses, generating the products OAS and CoASH. The rate-limiting step in the reaction at limiting l-serine levels is likely formation of the tetrahedral intermediate between serine and acetyl-CoA.     

Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD? as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 ? resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 ?, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >103-fold increases in V?/K(Lys)E(t) and V?/K(α-kg)E(t) (the double mutation gives >10?-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V?/K(Lys). A value of 2.0 was also observed for (D)(V?/K(Lys))(D?O) when the primary deuterium kinetic isotope effect was repeated in D?O, consistent with a rate-limiting hydride transfer step. A viscosity effect of 0.8 was observed on V?/K(Lys), indicating the solvent deuterium isotope effect resulted from stabilization of an enzyme form prior to hydride transfer. A small normal solvent isotope effect is observed on V, which decreases slightly when repeated with NADD, consistent with a contribution from product release to rate limitation. In addition, V?/K(Lys)E(t) is pH-independent, which is consistent with the loss of an acid-base catalyst and perturbation of the pK(a) of the second catalytic group to a higher pH, likely a result of a change in the overall charge of the active site. The primary deuterium kinetic isotope effect for H96Q, measured in H?O or D?O, is within error equal to 1. A solvent deuterium isotope effect of 2.4 is observed with NADH or NADD as the dinucleotide substrate. Data suggest rate-limiting imine formation, consistent with the proposed role of H96 in protonating the leaving hydroxyl as the imine is formed. The pH-rate profile for V?/K(Lys)E(t) exhibits the pK(a) for K77, perturbed to a value of ～9, which must be unprotonated to accept a proton from the ε-amine of the substrate Lys so that it can act as a nucleophile. Overall, data are consistent with a role for K77 acting as the base that accepts a proton from the ε-amine of the substrate lysine prior to nucleophilic attack on the α-oxo group of α-ketoglutarate, and finally donating a proton to the imine nitrogen as it is reduced to give saccharopine. In addition, data indicate a role for H96 acting as a general acid-base catalyst in the formation of the imine between the ε-amine of lysine and the α-oxo group of α-ketoglutarate.     

Full-length and N-TIMP-3 display equal inhibitory activities toward TNF-alpha convertase

We previously reported that tumor necrosis factor-alpha converting enzyme (TACE) was specifically inhibited by TIMP-3 but not TIMP-1, -2, and -4. Further mutagenesis studies showed that the N-terminal domain of TIMP-3 (N-TIMP-3) retained full inhibitory activity towards TACE. Full-length TIMP-3 and N-TIMP-3 exhibited indistinguishable values for the association rate constant and inhibitory affinity constant for the active catalytic domain of TACE (k(on) approximately 10(5) M(-1) s(-1) and K(app)(i) approximately 0.20 nM). Moreover, their k(on) (approximately 10(4) M(-1) s(-1)) and K(app)(i) (approximately 1.0 nM) values with a longer form of TACE (which encompasses the complete ectodomain including disintegrin, EGF and Crambin-like domains) were also shown to be similar. Detailed kinetic analyses indicated that TIMP-3 associated more quickly and with tighter final binding with TACE devoid of these C-terminal domains. We conclude that, unlike the interaction between many MMPs and TIMPs, the C-terminal domains of TIMP-3 and TACE are not essential in the formation of a tight binary complex.