Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile. Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide. The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose. A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised. For the wild-type enzyme, the value of the Br?nsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups. Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining. The Br?nsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively). These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1). Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism.     

Mechanistic requirements for the efficient enzyme-catalyzed hydrolysis of thiosialosides

The sialidase from Micromonospora viridifaciens has been found to catalyze the hydrolysis of aryl 2-thio-alpha-D-sialosides with remarkable efficiency: the first- and second-order rate constants, kcat and kcat/Km, for the enzyme-catalyzed hydrolysis of PNP-S-NeuAc are 196 +/- 5 s(-1) and (6.7 +/- 0.7) x 10(5) M(-1) s(-1), respectively. A reagent panel of eight aryl 2-thio-alpha-D-sialosides was synthesized and used to probe the mechanism for the M. viridifaciens sialidase-catalyzed hydrolysis reaction. In the case of the wild-type enzyme, the derived Br?nsted parameters (beta(lg)) on kcat and kcat/Km are -0.83 +/- 0.11 and -1.27 +/- 0.17 for substrates with thiophenoxide leaving groups of pKa values > or = 4.5. For the general-acid mutant, D92G, the derived beta(lg) value on kcat for the same set of leaving groups is -0.82 +/- 0.12. When the conjugate acid of the departing thiophenol was < or = 4.5, the derived Br?nsted slopes for both the wild-type and the D92G mutant sialidase were close to zero. In contrast, the nucleophilic mutant, Y370G, did not display a similar break in the Br?nsted plots, and the corresponding values for beta(lg), for the three most reactive aryl 2-thiosialosides, on kcat and kcat/Km are -0.76 +/- 0.28 and -0.84 +/- 0.04, respectively. Thus, for the Y370G enzyme glycosidic C-S bond cleavage is rate-determining for both kcat and kcat/Km, whereas, for both the wild-type and D92G mutant enzymes, the presented data are consistent with a change in rate-determining step from glycosidic C-S bond cleavage for substrates in which the pKa of the conjugate acid of the leaving group is > or = 4.5, to either deglycosylation (kcat) or a conformational change that occurs prior to C-S bond cleavage (kcat/Km) for the most activated leaving groups. Thus, the enzyme-catalyzed hydrolysis of 2-thiosialosides is strongly catalyzed by the nucleophilic tyrosine residue, yet the C-S bond cleavage does not require the conserved aspartic acid residue (D92) to act as a general-acid catalyst.     

Photoaffinity labeling of tubulin with (2-nitro-4-azidophenyl)deacetylcolchicine: direct evidence for two colchicine binding sites

A new photoaffinity analogue of colchicine, (2-nitro-4-azidophenyl)deacetylcolchicine (NAPDAC), bound to two classes of sites on bovine renal tubulin and photolabeled both the alpha- and beta-subunits. The apparent Ki for the photoaffinity analogue was 1.40 +/- 0.17 microM (mean +/- SD, n = 3) as measured by competition with [3H] colchicine. Values of the apparent KdS for the two sites, as measured by the direct binding of the [3H]NAPDAC to tubulin, were 0.48 +/- 0.11 microM and 11.6 +/- 3.5 microM (mean +/- SD, n = 6), and the corresponding stoichiometries of binding of the two sites were 0.25 +/- 0.06 and 1.3 +/- 0.4 mol/mol of tubulin (mean +/- SD, n = 6). NAPDAC was a potent inhibitor of microtubule formation as detected by electron microscopy. When tubulin was photolabeled with NAPDAC at 25 degrees C, 15 +/- 3 mol % (mean +/- SD, n = 6) of the [3H]NAPDAC was covalently bound to the alpha-subunit, and 67 +/- 9 mol % (mean +/- SD, n = 6) was covalently bound to the beta-subunit. Since NAPDAC is a mixture of two interconvertible diastereomers, the photoincorporation of each was also examined. One diastereomer photolabeled both alpha- and beta-tubulin; however, the other did not significantly photolabel either subunit. Tubulin photolabeled with NAPDAC (1:1 mole ratio) exhibited a 23% decrease in colchicine binding. Preblocking and prephotolysis experiments with colchicine, NAPDAC, or ANPAH-CLC [Williams et al. (1985) J. Biol. Chem. 260, 13794-13802] provided evidence for conformational changes in tubulin upon colchicine binding. Peptide maps of [3H]NAPDAC-labeled alpha- and beta-tubulin, using Staphylococcus aureus V8 protease, demonstrated the presence of NAPDAC in one peptide of the alpha-subunit and in five peptides of the beta-subunit as detected by autoradiography. NAPDAC provides the first direct evidence for two colchicine binding sites on tubulin.     

Catalytic competence, a new criterion for affinity labeling. Demonstration of the reversible enzymatic interconversion of estrone and estradiol-17 beta covalently bound to human placental estradiol-17 beta dehydrogenase.

Human placental estradiol-17beta dehydrogenase is rapidly inactivated upon treatment with 3-bromoacetoxyestrone. Pseudo-first order kinetic data are obtained and inactivation is accompanied by incorporation of 1 mol of 3-acetoxyestrone/mol of subunit (Mr =34,000). Treatment of the inactivated enzyme with (4S)-[4-2H]DPNH results in the formation of covalently bound [17alpha-2H]estradiol-17beta, which can be released by hydrolysis and identified by gas chromatography-mass sepctrometry. When (4R)-[4-2H]DPNH was used, deuterium was not transferred. Thus, the normal stereochemistry of hydridetransfer is preserved for both partners. After treatment with p-mercuribenzoate, affinity-labeled estradiol-17beta dehyrogenase is no longer able to caralyze reduction its covalently bound estrone; in the presence of DPNH and native enzyme, however, reduction occurs, demonstrating that affinity-labeled enzyme can itself serve as subtrate for native estradiol-17beta dehydrogenase. The reversible enzymatic interconversion of covalently bound estrone was demonstrated using a transhydrogenase assay. The ability of an enzyme to catalyze its normal reaction with a covalently bound substrate is termed catalytic competence, and is considered to be a new criterion for affinity labeling.     

Kinetic isotope effect studies on milk xanthine oxidase and on chicken liver xanthine dehydrogenase

The effect of isotopic substitution of the 8-H of xanthine (with 2H and 3H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in H/T(V/K) values was observed between xanthine oxidase (3.59 +/- 0.1) and xanthine dehydrogenase (3.60 +/- 0.09). Xanthine dehydrogenase exhibited a larger T/D(V/K) value (0.616 +/- 0.028) than that observed for xanthine oxidase (0.551 +/- 0.016). Observed H/T(V/K) values for either enzyme are less than those H/T(V/K) values calculated with D/T(V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8-3H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (Cr) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect (Dk) for xanthine oxidation is calculated to be 7.4 +/- 0.7 for xanthine oxidase and 4.2 +/- 0.2 for xanthine dehydrogenase. By use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be approximately 75-fold faster than kcat for xanthine oxidase and approximately 10-fold faster than kcat for xanthine dehydrogenase. This calculated rate is consistent with data obtained by rapid-quench experiments with XO. A stoichiometry of 1.0 +/- 0.3 mol of uric acid/mol of functional enzyme is formed within the mixing time of the instrument (5-10 ms). The kinetic isotope effect data also permitted the calculation of the Kd values [Klinman, J. P., & Mathews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] for substrate dissociation, including all reversible steps prior to C-H bond cleavage. Values calculated for each enzyme (Kd = 120 microM) were found to be identical within experimental uncertainty.     

Purification and properties of an iron-sulfur-containing and pyridoxal-phosphate-independent L-serine dehydratase from Peptostreptococcus asaccharolyticus

L-Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on L-glutamate or L-serine. The enzyme was highly specific for L-serine with the lowest Km = 0.8 mM ever reported for an L-serine dehydratase. L-Threonine (Km = 22 mM) was the only other substrate. V/Km for L-serine was 500 times higher than that for L-threonine. L-Cysteine was the best inhibitor (Ki = 0.3 mM, competitive towards L-serine). The enzyme was purified 400-fold to homogeneity under anaerobic conditions (specific activity 6 mukat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (alpha, 30 kDa; beta, 25 kDa). The molecular mass of the native enzyme was estimated as 200 +/- 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine (5 mM each). However, by exposure to air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 +/- 0.2 mol Fe and 5.6 +/- 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe-S] center. The enzyme was successfully applied to measure L-serine concentrations in bacterial media and in human sera.     

Deuterated delta 7-cholestenol analogues as mechanistic probes for wild-type and mutated delta 7-sterol-C5(6)-desaturase

Deuterium-labeled 5alpha-cholest-7-en-3beta-ol (1) bearing one or two deuteriums at the C-5alpha and (or) C-6alpha positions was synthesized in high isotopic and chiral purity. These compounds were used as substrates with the microsomal wild-type Zea mays and recombinant Arabidopsis thaliana Delta(7)-sterol-C5(6)-desaturases (5-DES) to probe directly the stereochemistry and the mechanism of the enzymatic reaction. Clearly, in the conversion of 1 by both 5-DESs, the 6alpha-hydrogen is removed. [6alpha-(2)H]-5alpha-Cholest-7-en-3beta-ol shows an intermolecular deuterium kinetic isotope effect (DKIE) on V and V/K, (D6)V = 2.6+/-0.3, (D6)V/K = 2.4+/-0.1; and (D6)V = 2.3 +/-0.3, (D6)V/K = 2.3+/-0.2 for the Zea mays and A. thaliana wild-type 5-DES, respectively. In contrast, negligible or minor isotope effects, (D5)V = 0.99+/-0.04, (D5)V/K = 0.91+/-0.08; and (D5)V = 0.93 +/-0.06, (D5)V/K = 0.96+/-0.04, respectively, were observed with [5alpha-(2)H]-cholest-7-en-3beta-ol. The observed pattern of isotope effects strongly suggests that the plant 5-DES initiates oxidation by cleavage of the chemically activated C6alpha-H bond, a step which appears to be partially rate-limiting in the desaturation process. Cleavage of the C5-H bond has a negligible isotope effect, indicating that the desaturation involves asynchronous scission of the two C-H bonds at C5 and C6. We showed previously [Taton, M., et al. (2000) Biochemistry 39, 701] that threonine 114 was not essential to maintaining desaturase activity, although V/K values for mutant T114I and T114S were respectively 10-fold lower and 4-fold higher than that of the native 5-DES. In this study, we combined variation in enzyme structure and DKIE studies and showed that (D6)V and (D6)V/K increased respectively to 3.8+/-0.3 and 3.8+/-0.4 in mutant T114I and decreased respectively to 1.6+/-0.4 and 1.7+/- 0.1 in mutant T114S. The data suggest that the conserved hydroxyl function at position 114 in the ERG3 family makes the abstraction of the 6alpha-hydrogen atom substantially less rate-limiting during the 5-DES reaction. Based on the data, a tentative mechanism for the desaturation of cholest-7-en-3beta-ol is proposed.     

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transition of hemoglobin between two tertiary conformations: determination of equilibrium and thermodynamic parameters from the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group

The equilibrium constant of the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group of hemoglobin decreases by 2 to 3 orders of magnitude between pH 5.6 and 9. The reaction is coupled to the ionizations of two groups on the protein. At 25 degrees C one group has a pK(a) of 5.31+/-0.2 when hemoglobin is in its (tertiary) r conformation, typified by the thiolate anion form of CysF9[93]beta; this changes to 7.73+/-0.4 in the (tertiary) t conformation, typified by the mixed disulfide form of the sulfhydryl. The second group ionizes with a pK(a) of 7.11+/-0.4 in the r conformation; this changes to 8.38+/-0.2 in the t conformation. K(rt), the equilibrium constant for the r<-->t isomerization process, is 0.22+/-0.06. The standard enthalpy and entropy changes for the isomerization are DeltaH(o)(rt)=24.2 kJ mol(-1) and DeltaS(o)(rt)=68.8 JK(-1)mol(-1), respectively.     

The effect of media pH and autocatalytic phosphorylation on the interaction of kinase phosphorylase with calmodulin

Using calmodulin covalently labeled with dansyl, the Ca2(+)-dependent interaction of phosphorylase kinase with calmodulin has been studied. It has been shown that at pH 6.8 the (alpha beta gamma delta) protomer of the enzyme binds 2.1 +/- 0.8 mol of calmodulin with Kd = (6.67 +/- 1.77).10(-8) M. The enzyme activation induced by the pH increase up to 8.2 does not affect the enzyme interaction with calmodulin [2.14 +/- 0.58 mol calmodulin per mol of (alpha beta gamma delta)]; Kd = (4.14 +/- 1.22).10(-8) M. However, the enzyme activation during its autocatalytic phosphorylation eliminates this effect practically completely.     

Structure and mechanism of action of an inverting mutant sialidase

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to small amino acids changes the mechanism of catalysis from retention of anomeric configuration to inversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682-12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3'-sialyllactose, the derived Br?nsted parameters (beta(lg)) on k(cat) and k(cat)/K(m) are -0.63 +/- 0.05 and -0.80 +/- 0.08, respectively. Thus, for the Y370G enzyme, glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant and wild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium alpha-d-N-acetylneuraminide (DHP-alphaNeu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactions proceeding via S(N)1 and S(N)2 mechanisms, respectively. The overall structure of the Y370G mutant sialidase active site is very similar to the previously reported wild-type structure [Gaskell, A., et al. (1995) Structure 3, 1197-1205], although removal of the tyrosine residue creates two significant changes to the active site. First, the anomeric oxygen atom of the hydrolysis product (beta-N-acetylneuraminic acid) and four water molecules bind in the large cavity created by the Y370G mutation. Second, the side chain of Asn310 moves to make a strong hydrogen bond to one of the bound water molecules.     

Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.     

Paramagnetic centers and acetyl-coenzyme A/CO exchange activity of carbon monoxide dehydrogenase from Methanothrix soehngenii.

Carbon monoxide (CO) dehydrogenase was purified, both aerobically and anaerobically, to apparent homogeneity from Methanothrix soehngenii. The enzyme contained 18 +/- 2 (n = 6) mol Fe/mol and 2.0 +/- 0.1 (n = 6) mol Ni/mol. Electron paramagnetic resonance (EPR) spectra of the aerobically purified CO dehydrogenase showed one sharp EPR signal at g = 2.014 with several characteristics of a [3Fe-4S]1+ cluster. The integrated intensity of this signal was low, 0.03 S = 1/2 spin/alpha beta dimer. The 3Fe spectrum was not affected by incubation with CO or acetyl-coenzyme A, but could be reduced by dithionite. The spectrum of the reduced, aerobically purified enzyme showed complex EPR spectra, which had several properties typical of two [4Fe-4S]1+ clusters, whose S = 1/2 spins weakly interacted by dipolar coupling. The integrated intensity was 0.1-0.2 spin/alpha beta dimer. The anaerobically isolated enzyme showed EPR spectra different from the reduced aerobically purified enzyme. Two major signals were apparent. One with g values of 2.05, 1.93 and 1.865, and an Em7.5 of -410 mV, which quantified to 0.9 S = 1/2 spin/alpha beta dimer. The other signal with g values of 1.997, 1.886 and 1.725, and an Em7.5 of -230 mV gave 0.1 spin/alpha beta dimer. When the enzyme was incubated with its physiological substrate acetyl-coenzyme A, these two major signals disappeared. Incubation of the enzyme under CO atmosphere resulted in a partial disappearance of the spectral component with g = 1.997, 1.886, 1.725. Acetyl-coenzyme A/CO exchange activity, 35 nmol.min-1.mg-1 protein, which corresponded to 7 mol CO exchanged min-1 mol-1 enzyme, could be detected in anaerobic enzyme preparations, but was absent in aerobic preparations. Carbon dioxide also exchanged with C-1 of acetyl-coenzyme A, but at a much lower rate than CO and to a much lower extent.     

The pH-dependent binding of NADH and subsequent enzyme isomerization of human liver beta 3 beta 3 alcohol dehydrogenase

Human class I beta 3 beta 3 is one of the alcohol dehydrogenase dimers that catalyzes the reversible oxidation of ethanol. The beta 3 subunit has a Cys substitution for Arg-369 (beta 369C) in the coenzyme-binding site of the beta1 subunit. Kinetic studies have demonstrated that this natural mutation in the coenzyme-binding site decreases affinity for NAD+ and NADH. Structural studies suggest that the enzyme isomerizes from an open to closed form with coenzyme binding. However, the extent to which this isomerization limits catalysis is not known. In this study, stopped-flow kinetics were used from pH 6 to 9 with recombinant beta 369C to evaluate rate-limiting steps in coenzyme association and catalysis. Association rates of NADH approached an apparent zero-order rate with increasing NADH concentrations at pH 7.5 (42 +/- 1 s-1). This observation is consistent with an NADH-induced isomerization of the enzyme from an open to closed conformation. The pH dependence of apparent zero-order rate constants fit best a model in which a single ionization limits diminishing rates (pKa = 7.2 +/- 0.1), and coincided with Vmax values for acetaldehyde reduction. This indicates that NADH-induced isomerization to a closed conformation may be rate-limiting for acetaldehyde reduction. The pH dependence of equilibrium NADH-binding constants fits best a model in which a single ionization leads to a loss in NADH affinity (pKa = 8.1 +/- 0. 2). Rate constants for isomerization from a closed to open conformation were also calculated, and these values coincided with Vmax for ethanol oxidation above pH 7.5. This suggests that NADH-induced isomerization of beta 369C from a closed to open conformation is rate-limiting for ethanol oxidation above pH 7.5.     

Mutation of Arg-166 of alkaline phosphatase alters the thio effect but not the transition state for phosphoryl transfer. Implications for the interpretation of thio effects in reactions of phosphatases

It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0. 06) are the same. The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.     

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Br?nsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.     

The use of [3H]aniline to identify the essential carboxyl group in the bovine mitochondrial F1-ATPase that reacts with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline

The inactivation of the bovine heart mitochondrial F1-ATPase with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of [3H]aniline at pH 7.0 led to the covalent incorporation of 3H into the enzyme. When the ATPase was inactivated by 94% with 0.9 mM EEDQ in the presence of 3.6 mM [3H]aniline in a large-scale experiment in which the protein concentration was 21 mg/ml, 4.2 mol [3H]anilide were formed per mol enzyme, of which 0.35 mol was incorporated per mol of the alpha subunit and 1.0 mol was incorporated per mol of the beta subunit. Examination of a tryptic digest of the isolated alpha subunit revealed that the majority of the 3H was contained in a single tryptic peptide, which, when purified, was shown to contain the [3H]anilide of a glutamic acid residue which corresponds to alpha-Glu-402 of the Escherichia coli F1-ATPase. This residue was labeled to the extent of about 1.0 mol/mol enzyme. Analysis of tryptic peptides purified from the isolated beta subunit showed that 0.8 and 1.5 mol, respectively, of the [3H]anilides of beta-Glu-341 and beta-Glu-199 were formed per mol MF1 during the inactivation of the enzyme at 21 mg/ml. When the ATPase was inactivated by 90% at a protein concentration of 1.7 mg/ml by 0.9 mM EEDQ in the presence of 1.7 mM [3H]aniline, 3.1 mol [3H]anilide were formed per mol enzyme. From the analysis of the radioactive peptides purified from a tryptic digest of the labeled ATPase from this experiment it was estimated that 0.7 mol of the [3H]anilide of alpha-Glu-402, 0.3 mol of the [3H]anilide of beta-Glu-341, and 1.5 mol of the [3H]anilide of beta-Glu-199 were formed per mol F1-ATPase. Since beta-Glu-199 is labeled to the same extent in the two experiments while alpha-Glu-402 and beta-Glu-341 were not, suggests that the modification of beta-Glu-199 is responsible for inactivation of the enzyme by EEDQ.     

Inhibition Effects in the Hydrolysis Reactions of Esters and Peptides Catalyzed by Carboxypeptidase A: An Example of Cooperative Binding Effects with a Monomeric Enzyme

N-benzoyl-L-phenylalanyl-L-phenylalanine is an excellent peptide substrate for carboxy-peptidase A; at 30 degrees C and pH 7.5, K(m) is 2.6 x 10(-5) M while k(cat) is 177 s(-1) (k(cat)/K(m) = 6.8 x 10(6) M(-1) s(-1)). Indole-3-acetic acid is a noncompetitive or mixed inhibitor towards the peptide and toward hippuryl-L-phenylalanine; plots of E/V vs [Inhibitor] are linear. N-Benzoyl-L-phenylalanine is a competitive inhibitor of peptide hydrolysis, and plots of E/V vs [Inhibitor] are again linear. One molecule of inhibitor binds per active site, and these inhibitors bind in different sites. At constant peptide substrate concentration and a series of constant concentrations of indole-3-acetic acid, plots of E/V vs the concentration of N-benzoyl-L-phenylalanine are linear and intersect behind the E/V axis and above the [Inhibitor] axis. This shows that both inhibitors can bind simultaneously and that binding of one facilitates the binding of the other (beta = 0.18). Employing the ester substrate hippuryl-DL,beta-phenyllactate, the same type of behavior is observed in the reverse sense; N-benzoyl-L-phenylalanine is a linear noncompetitive inhibitor and indole-3-acetic acid is a linear competitive inhibitor. Again the two inhibitor plot is linear and intersects above the [Inhibitor] axis (beta = 0.12). Previous X-ray crystallographic studies have indicated that indole-3-acetic acid binds in the hydrophobic pocket of the S'(1) site, while N-benzoyl-L-phenylalanine binds in the S(1)-S(2) site. The product complex for hydrolysis of N-benzoyl-L-phenylalanyl-L-phenylalanine (phenylalanine + N-benzoyl-L-phenylalanine) occupies both of these sites. However, the present work shows that the peptide substrate does not bind to the enzyme at pH 7.5 so as to be competitive with indole-3-acetic acid. The binding sites may be formed via conformational changes induced or stabilized by substrate and product binding. Copyright 2000 Academic Press.