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M F Dunn

Publications and source records attributed to M F Dunn.

At least 37 records · Page 2Linked to original sources
PubMed1996

Substitution of pyridoxal 5'-phosphate in the O-acetylserine sulfhydrylase from Salmonella typhimurium by cofactor analogs provides a test of the mechanism proposed for formation of the alpha-aminoacrylate intermediate.

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final step in the de novo synthesis of L-cysteine in Salmonella typhimurium. Complementary cofactor mutagenesis in which the active site PLP is substituted with cofactor analogs is used to test the mechanism proposed for the OASS. Data obtained with the pyridoxal 5'-deoxymethylenephosphonate-substituted enzyme suggest that the binding of OAS as it forms the external Schiff base is such that the acetate side chain is properly positioned for elimination (orthogonal to the developing alpha,beta-double bond) only about 1% of the time. Data support the assignment of an enzyme group with a pK of 6.7 that interacts with the acetyl side chain, maintaining it orthogonal to the developing alpha,beta-double bond. Similar studies of the 2'-methylpyridoxal 5'-phosphate-substituted enzyme suggest that, although the mechanism is identical to that catalyzed by native OASS, the reaction coordinate for alpha-proton abstraction may be decreased compared with that observed for the native enzyme.

Alanine↗
PubMed1996

Evidence of a low-barrier hydrogen bond in the tryptophan synthase catalytic mechanism.

In the absence of other substrates, L-Ser reacts rapidly with the tryptophan synthase alpha 2 beta 2 bienzyme from Salmonella typhimurium at pH 7.8 and 25 degrees C to give an equilibrating mixture of species dominated by comparable amounts of the L-Ser external aldimine Schiff base, E(Aex1), and the alpha-aminoacrylate Schiff base, E(A-A). The D-isomer of Ser is unreactive toward alpha 2 beta 2, and therefore, D,L-Ser can be used in place of L-Ser for investigations of catalytic mechanism. Due to the equilibrium isotope effect, when alpha-2H-D,L-Ser is substituted for alpha-1H-D,L-Ser, the position of equilibrium is shifted in favor of E(Aex1). On a much slower time scale, the 2H sample undergoes the exchange of enzyme bound 2H for the 1H of solvent water and is converted to a distribution of E(Aex1) and E(A-A) identical to that obtained with the 1H sample. This slow exchange indicates that the proton abstracted from the alpha-carbon of E(Aex1) is sequestered within a solvent-excluded site in E(A-A). Analysis of the UV/vis spectra gave an isotope effect on the equilibrium distribution of E(Aex1) and E(A-A) of KH/KD = 1.80 +/- 0.18. This large equilibrium isotope effect is the consequence of an unusual isotope fractionation factor of 0.62 for the residue which functions as the base to deprotonate and protonate the alpha-carbon proton in E(Aex1). A fractionation factor of 0.62 qualifies as evidence for the involvement of a low-barrier H-bond (LBHB) in this equilibration. Since this effect arises from abstraction of the alpha-proton from E(Aex1), the LBHB must be associated with the E(A-A) species. In contrast to weak H-bonds with energies of 3-12 kcal/mol, LBHBs are proposed to exhibit energies in the 12-24 kcal/mol range [Frey, P.A., Whitt, S.A., & Tobin, J. B. (1994) Science 264, 1927-1930]. Possible roles for this LBHB both in the chemical mechanism and in the stabilization of the closed conformation of E(A-A) are discussed.

Binding Sites↗
PubMed1996

Kinetic isotope effects as a probe of the beta-elimination reaction catalyzed by O-acetylserine sulfhydrylase.

Primary and alpha-secondary deuterium kinetic isotope effects have been measured for the O-acetylserine sulfhydrylase from Salmonella typhimurium using both steady-state and single-wavelength stopped-flow studies. Data suggest an asymmetric transition state for alpha-proton abstraction by the active site lysine and the elimination of the acetyl group of O-acetyl-L-serine (OAS) to form the alpha-aminoacrylate intermediate. The value of D(V/KOAS) using OAS-2-d is dependent on pH from 5.8 to 7.0 with independent values of 2.8 and 1.7 estimated at low and high pH, respectively. Thus, OAS is sticky, and a value of 1.5 is calculated for the forward commitment to catalysis, indicating that the OAS external Schiff base preferentially partitions toward the alpha-aminoacrylate intermediate compared to OAS being released from enzyme. The intrinsic primary deuterium isotope effect determined from single-wavelength stopped-flow studies of alpha-proton abstraction by the active site lysine is about 2.0. D(V/KOAS) and T(V/KOAS) were determined as 2.6 +/- 0.1 and 4.2 +/- 0.2 at pH 6.1, respectively, giving a calculated intrinsic deuterium isotope effect of 3.3 +/- 0.9, consistent with the D(V/KOAS) obtained from steady-state studies at low pH. The alpha-secondary deuterium kinetic isotope effect using OAS-3,3-d2 is 1.11 +/- 0.06 obtained by direct comparison of initial velocities and 1.2 obtained by single-wavelength stopped-flow experiments. Data can be compared to a value of 1.81 +/- 0.04 using OAS-3,3-d2 for alpha-DKeq for the first half-reaction.

Catalysis↗
PubMed1996

Formation of the alpha-aminoacrylate immediate limits the overall reaction catalyzed by O-acetylserine sulfhydrylase.

O-Acetylserine sulfhydrylase-A (OASS-A) catalyzes the final step in the synthesis of L-cysteine, viz., the beta-substitution of acetate in O-acetyl-L-serine (OAS) by sulfide via a ping-pong kinetic mechanism . Rapid-scanning stopped-flow and single-wavelength absorbance and fluorescence stopped-flow experiments were carried out to obtain information on the location and amount of limitation of rate-determining steps for the overall reaction and the individual half-reactions of OASS-A. The first half-reaction, conversion of OAS to the alpha-aminoacrylate intermediate and acetate, is rate-limiting for the overall reaction catalyzed by OASS-A. No intermeidates are detected within the second half-reaction, and thus rate constants for all steps must be > or = 1000s-1 at the lowest sulfide concentration used. Within the first half reaction, formation of the extrernal Schiff base (Kassociation = 0.2 mM-1) is observed in the first milliseconds, followed by its slower conversion to the alpha-aminocacrylate intermediate with a rate constant of 300 s-1, close to the value of 130 s-1 obtained for V/Et [Tai, C.H., Nalabolu, S.R., Jacobson, T.M., Minter D.E., & Cook, P.F. (1993) Biochemistry 32, 6433-6442]. Addition of L-cysterine ot OASS-A results in a rapid formation of the external Schiff base (Kassociation = 6.7 mM-1), followed by transient formation of the alpha-aminoacylate intermediate with a slightly lower rate (70-100 s-1) compared to OAS. The alpha-aminoacrylate intermediate decays to generate a species absorbing maximally at 418 nm, resulting from attack of the cysteine thiol to give ether in external Schiff base linkage with the active site PLP.

Alanine↗
PubMed1996

beta-Site covalent reactions trigger transitions between open and closed conformations of the tryptophan synthase bienzyme complex.

The tryptophan synthase bienzyme complex (alpha2beta2) from Salmonella tryphimurium catalyzes the final steps in the biosynthesis of L-Trp. To investigate the roles played by conformational change in tryptopthan synthase catalysis, the fluorophore 8-anilino-1-naphthalensulfonate (ANS) is used to identify conformational states. The binding of ANS to the alpha2beta2 bienzyme complex is accompanied by a dramatic enhancement of ANS fluorescence and a shift of the emission maximum from 520 to 482 nm. The ANS binding isotherm is biphasic and consists of a class of moderately high-affinity, noninteracting sites with a stoichiometry of 1 site/alpha beta dimeric unit (Kd' = 62 + or - 15 micrometer) and a much weaker set of non-specific interactions with K'd>1mM. Our findings show that the affinity of the enzyme for ANS is strongly decreased (> 10-fold) by interactions at two loci 30 angstroms apart: (i) the binding of the alpha-site ligands, 3-indole-D-glycerol 3'-phosphate or alpha-glycerol phosphate (GP) or (ii) reaction at the beta-subunit to form either the alpha-aminoacrylate Schiff base, E(A-A), or quinonoid species, E(Q). In contrast, formation of the L-Ser and L-Trp external aldimines E(Aex1) and E(Aex2) at the beta-site causes a 2-3 fold decrease in the affinity of the enzyme for ANS. The combination of E(A-A)or E(Q) with GP brings about almost complete displacement of ANS, indicating that these interactions drive a conformation change in alphabeta subunit pairs which prevents the binding of ANS. These results are consistent with a model which postulates that alphabeta subunit pairs undergo ligand-mediated transitions between open and closed conformations during the catalytic cycle. Consistent with the kinetic data showing that binding of alpha-site ligands increases the affinity of the beta site for L-Ser and that formation of E(A-A) activates the alpha reaction [Brzović, P. S., Ngo, K., & Dunn, M. F. (1992) Biochemistry 31, 3831-3839], while mutations in alpha subunit loops 2 and 6 prevent the ligand- mediated transition to a closed structure [Brzović, P.S., Hyde, C.C., Miles, E.W., & Dunn, M.F. (1993) Biochemistry 32, 10404-10413], we conclude that reciprocal ligand-mediated allosteric interactions between the heterologous subunits promote conformational transitions between open and closed structures in alphabeta subunit pairs which function to coordinate catalytic activities and facilitate the channeling of indole between the two catalytic sites.

Anilino Naphthalenesulfonates↗
PubMed1996

Allosteric regulation of tryptophan synthase: effects of pH, temperature, and alpha-subunit ligands on the equilibrium distribution of pyridoxal 5'-phosphate-L-serine intermediates.

The equilibrium distribution of catalytic intermediates formed in the reaction of L-serine with the tryptophan synthase alpha 2 beta 2-complex from Salmonella typhimurium has been investigated by absorption and fluorescence spectroscopy as a function of pH, temperature, and alpha-subunit ligands. The novel result of this study is that the equilibrium between the two major catalytic species, the external aldimine and the alpha-aminoacrylate, is modulated by the ionization of two groups with apparent pK values of 7.8 +/- 0.3 and 10.3 +/- 0.2. Protonation of these groups stabilizes the alpha-aminoacrylate Schiff base by an estimated 100-fold with respect to the external aldimine. Furthermore, the formation of the alpha-aminoacrylate from the external aldimine is an endothermic process. Temperature slightly affects the apparent pK values but remarkably influences the amplitude of the phase associated with the ionization of each group. At 20 degrees C, each phase accounts for nearly half of the titration. Since the isolated beta 2-dimer does not exhibit a pH-dependent distribution of intermediates, the alpha-beta-subunit interactions seem critical to the onset of this functional property of the beta-subunit. The modulation of intersubunit interactions by the alpha-subunit ligands DL-alpha-glycerol 3-phosphate and phosphate leads to significant changes in the pH-dependent distribution of intermediates. At saturating concentrations of either of these alpha-subunit ligands, the alpha-aminoacrylate Schiff base is the predominant species over a wide pH range while the apparent pK values of the groups that control the equilibrium are not significantly affected. The pH-dependent interconversion of catalytic intermediates here reported has not been previously detected because phosphate buffers have usually been employed in the studies of this enzyme. Our findings are discussed in the light of a model in which specific protein conformations are associated with the external aldimine and the alpha-aminoacrylate Schiff bases, the latter being stabilized by temperature, protons, and alpha-subunit ligands.

Allosteric Regulation↗
PubMed1996

Spectroscopic evidence for preexisting T- and R-state insulin hexamer conformations.

The insulin hexamer is an allosteric protein exhibiting both positive and negative cooperative homotropic interactions and positive cooperative heterotropic interactions (C. R. Bloom et al., J. Mol. Biol. 245, 324-330, 1995). In this study, detailed spectroscopic analyses of the UV/Vis absorbance spectra of the Co(II)-substituted human insulin hexamer and the 1H NMR spectra of the Zn(II)-substituted hexamer have been carried out under a variety of ligation conditions to test the applicability of the sequential (KNF) and the half-site reactivity (SMB) models for allostery. Through spectral decomposition of the characteristic d-->d transitions of the octahedral Co(II)-T-state and tetrahedral Co(II)-R-state species, and analysis of the 1H NMR spectra of T- and R-state species, these studies establish the presence of preexisting T- and R-state protein conformations in the absence of ligands for the phenolic pockets. The demonstration of preexisting R-state species with unoccupied sites is incompatible with the principles upon which the KNF model is based. However, the SMB model requires preexisting T- and R-states. This feature, and the symmetry constraints of the SMB model make it appropriate for describing the allosteric properties of the insulin hexamer.

Allosteric Regulation↗
PubMed1996

Pyruvate carboxylase from Rhizobium etli: mutant characterization, nucleotide sequence, and physiological role.

Pyruvate carboxylase (PYC), a biotin-dependent enzyme which catalyzes the conversion of pyruvate to oxaloacetate, was hypothesized to play an important anaplerotic role in the growth of Rhizobium etli during serial subcultivation in minimal media containing succinate (S. Encarnación, M. Dunn, K. Willms, and J. Mora, J. Bacteriol. 177:3058-3066, 1995). R. etli and R. tropici pyc::Tn5-mob mutants were selected for their inability to grow in minimal medium with pyruvate as a sole carbon source. During serial subcultivation in minimal medium containing 30 mM succinate, the R. etli parent and pyc mutant strains exhibited similar decreases in growth rate with each subculture. Supplementation of the medium with biotin prevented the growth decrease of the parent but not the mutant strain, indicating that PYC was necessary for the growth of R. etli under these conditions. The R. tropici pyc mutant grew normally in subcultures regardless of biotin supplementation. The symbiotic phenotypes of the pyc mutants from both species were similar to those of the parent strains. The R. etli pyc was cloned, sequenced, and found to encode a 126-kDa protein of 1,154 amino acids. The deduced amino acid sequence is highly homologous to other PYC sequences, and the catalytic domains involved in carboxylation, pyruvate binding, and biotinylation are conserved. The sequence and biochemical data show that the R. etli PYC is a member of the alpha4, homotetrameric, acetyl coenzyme A-activated class of PYCs.

Amino Acid Sequence↗
PubMed1995

Monovalent metal ions play an essential role in catalysis and intersubunit communication in the tryptophan synthase bienzyme complex.

This investigation shows that the alpha 2 beta 2 tryptophan synthase bienzyme complex from Salmonella typhimurium is subject to monovalent metal ion activation. The effects of the monovalent metal ions Na+ and K+ were investigated using rapid scanning stopped-flow (RSSF), single-wavelength stopped-flow (SWSF), and steady-state techniques. RSSF measurements of individual steps in the reaction of L-serine and indole to give L-trytophan (the beta-reaction) as well as the reaction of 3-indole-D-glycerol 3'-phosphate (IGP) with L-serine (the alpha beta-reaction) demonstrate that monovalent metal ions such as Na+ and K+ change the distribution of intermediates in both the transient and steady states. Therefore the metal ion effect alters relative ground-state energies and the relative positions of ground- and transition-state energies. The RSSF spectra and SWSF time courses show that the turnover of indole is significantly reduced in the absence of either Na+ or K+. The alpha-aminoacrylate Schiff base species, E(A-A), is in a less active state in the absence of monovalent metal ions. Na+ decreases the steady-state rate of IGP cleavage (the alpha-reaction) to about 30% of the value obtained in the absence of metal ions. Steady-state investigations show that in the absence of monovalent metal ions the alpha- and alpha beta-reactions have the same activity. Na+ binding gives a 30-fold stimulation of the alpha-reaction when the beta-site is in the E(A-A) form.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Allosteric linkages between beta-site covalent transformations and alpha-site activation and deactivation in the tryptophan synthase bienzyme complex.

This work examines two aspects of the catalytic mechanism and allosteric regulation of the tryptophan synthase bienzyme complex from Salmonella typhimurium: (a) the chemical mechanism by which indole and other nucleophiles react with the enzyme-bound alpha-aminoacrylate Schiff base intermediate, E(A-A), to form quinonoidal intermediates, E(Q), and (b) the effects of covalent transformations at the beta-site on the catalytic activity of the alpha-site. Transient kinetic studies in combination with alpha-secondary deuterium isotope effects are undertaken to determine the mechanism of nucleophile addition to E(A-A). These studies establish that nucleophilic attack is best described by a two-step reaction sequence consisting of a binding step that is followed by Michael addition to the conjugated double bond of E(A-A). Analysis of isotope effects suggests that the transition state for indole addition gives an E(A-A) beta-carbon that resembles an sp3 center, while the stronger nucleophiles, indoline and beta-mercaptoethanol, have transition states that appear to more closely resemble an sp2 beta-carbon. The effects of beta-site covalent transformations on alpha-site catalysis were studied using quasi-stable beta-site intermediates and the alpha-site substrate analogue 3-[6-nitroindole]-D-glycerol 3'-phosphate (6-nitro-IGP). It was found that the cleavage of 6-nitro-IGP is strongly activated by the formation of E(A-A) and various E(Q) species at the beta-site but not by external aldimine species. Therefore, we conclude that the conversion of the L-Ser external aldimine to E(A-A) is the beta-site process which activates the alpha-site, while conversion of E(Q) to the L-Trp external aldimine triggers deactivation of the alpha-site. These findings are discussed within the context of allosteric regulation of substrate channeling in tryptophan synthase catalysis.

Allosteric Regulation↗
PubMed1995

Ligand binding to wild-type and E-B13Q mutant insulins: a three-state allosteric model system showing half-site reactivity.

By using ultra-violet and visible absorbance in conjunction with high field 1H-nuclear magnetic resonance spectroscopy, the insulin hexamer has been shown to undergo two allosteric transitions in solution involving three allosteric states (T6<-->T3 R3<-->R6). A simple mathematical model consisting of four variables has been derived that quantitatively describes the complex homotropic and heterotropic interactions that modulate these allosteric transitions. The mutation of one residue, Glu-B13 to Gln, results in an unexpected change in the T3R3 to R6 equilibrium by a factor of 10(7).

Allosteric Site↗
PubMed1994

Structural asymmetry and half-site reactivity in the T to R allosteric transition of the insulin hexamer.

The zinc-insulin hexamer, the storage form of insulin in the pancreas, is an allosteric protein capable of undergoing transitions between three distinct conformational states, designated T6, T3R3, and R6, on the basis of their ligand binding properties, allosteric behavior, and pseudo point symmetries [Kaarsholm, N. C., Ko, H.-C., & Dunn, M. F. (1989) Biochemistry 28, 4427-4435]. The transition from the T-state to the R-state involves a coil-to-helix transition in residues 1-8 of the B-chain wherein the ring of PheB1 is displaced by approximately 30 A. This motion also is accompanied by small changes in the positions of A-chain residues and other B-chain residues. In this paper, one- and two-dimensional (COSY and NOESY) 1H NMR are used to characterize the ligand-induced T to R transitions of wild-type and EB13Q mutant human zinc-insulin hexamers and to make sequence-specific assignments of all resonances in the aromatic region of the R6 complex with resorcinol. The changes in the 1H NMR spectrum (at 500 and 600 MHz) that occur during the T to R transition provide specific signatures of the conformation change. Analysis of the dependence of these spectral changes for the phenol-induced transition as a function of the concentration of phenol establish (1) that the interconversion of T6 and R6 occurs via a third species assigned as T3R3 and (2) that the system shows both negative and positive cooperative allosteric behavior. One- and two-dimensional COSY and NOESY studies show that, in the absence of phenolic compounds, anions act as heterotropic effectors that shift the distribution of hexamer conformations in favor of the R-state with the order of effectiveness, SCN- > N3- >> I- >> Cl-. Analysis of one- and two-dimensional spectra indicate that with wild-type insulin, SCN- and N3- give T3R3 species, whereas the EB13Q mutant gives an R6 species. An allosteric model for the insulin T to R transition based on the structural asymmetry model [Seydoux, F., Malhotra, O. P., & Bernhard, S. A. (1974) CRC Crit. Rev. Biochem. 2, 227-257] is proposed that explains the negative and positive allosteric properties of the system, including the role of T3R3 and the action of homotropic and heterotropic effectors.

Allosteric Regulation↗
PubMed1994

Role of the proximal ligand in peroxidase catalysis. Crystallographic, kinetic, and spectral studies of cytochrome c peroxidase proximal ligand mutants.

The role of the proximal histidine ligand in peroxidase function was studied by replacing the His side chain in cytochrome c peroxidase with Gln, Glu, or Cys. In addition, a double mutant was prepared where His-175 is converted to Gln and the site of free radical formation in Compound I, Trp-191 (Sivaraja, M., Goodin, D.B., Smith, M., and Hoffman, B. M. (1989) Science 245, 738-740), is converted to Phe. With the exception of the His-175-->Cys mutant, the proximal ligand mutants retain high levels of enzyme activity. Stopped flow studies show that replacing the His ligand with Gln has only a modest effect on the rate of Compound I formation demonstrating that the precise nature of the proximal ligand is not important in achieving a high rate of peroxide O-O bond cleavage. The double mutant, His-175-->Gln/Trp-191-->Phe, also forms Compound I rapidly but the initial product formed is very likely a long-lived porphyrin pi cation radical that slowly converts to a species more closely resembling the heme oxyferryl center of wild type Compound I. The relevance of these studies to the cytochrome c peroxidase-cytochrome c electron transfer system are discussed.

Crystallography, X-Ray↗
PubMed1993

The allosteric transition of the insulin hexamer is modulated by homotropic and heterotropic interactions.

The allosteric behavior of the Co(II)-substituted insulin hexamer has been investigated using electronic spectroscopy to study the binding of different phenolic analogues and singly charged anions to effector sites on the protein. This work presents the first detailed, quantitative analysis of the ligand-induced T- to R-state allosteric transition of the insulin hexamer. Recent studies have established that there are two ligand binding processes which stabilize the R-state conformation of the Co(II)-substituted hexamer: the binding of cyclic organic molecules to the six protein pockets present in the Zn(II)-R6 insulin hexamer [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 594-596] and the coordination of singly charged anions to the His(B10) metal sites [Brader, M.L., Kaarsholm, N.C., Lee, W.K., & Dunn, M.F. (1991) Biochemistry 30, 6636-6645]. The R6 insulin hexamer is stabilized by heterotropic interactions between the hydrophobic protein pockets and the coordination sites of the His(B10)-bound metal ions. The binding studies with 4-hydroxybenzamide, m-cresol, resorcinol, and phenol presented herein show that, in the absence of inorganic anions, the 4-hydroxybenzamide-induced transition, with a Hill number of 2.8, is the most cooperative, followed by m-cresol, phenol, and resorcinol with Hill numbers of 1.8, 1.4, and 1.2, respectively. The relative effectiveness of these ligands in shifting the allosteric equilibrium in favor of the Co(II)-R6 hexamer was found to be resorcinol > phenol > 4-hydroxybenzamide > m-cresol.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1993

Characterization of the functional role of a flexible loop in the alpha-subunit of tryptophan synthase from Salmonella typhimurium by rapid-scanning, stopped-flow spectroscopy and site-directed mutagenesis.

The function of a flexible loop (loop 6) in the alpha-subunit from the tryptophan synthase alpha 2 beta 2 bienzyme complex has been investigated utilizing rapid-scanning (RSSF) and single-wavelength (SWSF) stopped-flow spectroscopies. Loop 6 is an extended sequence of residues which connects beta-strand 6 with alpha-helix 6 in the beta/alpha-barrel fold of the alpha-subunit. Substitution of Leu for Arg179 near the base of loop 6 does not significantly affect either the association of the alpha- and beta-subunits to form the bienzyme complex or the kinetics of the reaction of indole with L-serine (L-Ser) to form L-tryptophan (L-Trp), the process catalyzed by the wild-type beta-subunit [Kawasaki, H., Bauerle, R., Zon, G., Ahmed, S., & Miles, E. W. (1987) J. Biol. Chem. 262, 10678-10683]. However, the alpha-subunit-specific ligand glycerol phosphate (GP), which is an inhibitor of the wild-type beta-reaction, is a much less effective inhibitor of the alpha R179L-catalyzed beta-reaction. Equilibrium titration studies show that the affinity of GP for the alpha-site when either L-Ser or glycine is bound at the beta-site has been reduced by nearly 100- and 200-fold, respectively. SWSF analysis of the reaction of IGP and L-Ser to form L-Trp catalyzed by the bienzyme complex revealed a 15-fold reduction in the binding affinity of the alpha-site substrate 3-indole-D-glycerol 3'-phosphate (IGP) in the reaction catalyzed by the alpha R179L mutant as compared to the wild-type enzyme. These studies show that loop 6 is important both for ligand binding to the alpha-site and for the ligand-induced conformational transition of the alpha-subunit from an "open" to a "closed" structure. Modeling studies, based on extensive structural homology of the alpha-subunit with the glycolytic enzyme triosephosphate isomerase (TIM), predict that closure of loop 6 induced by ligand binding at the alpha-active site would effectively sequester the bound substrate from the solvent and trap indole, produced from the cleavage of IGP, within the confines of the bienzyme complex. This conformational transition would promote the diffusion of indole to the beta-active site via the interconnecting tunnel and would help ensure the close coordination of alpha- and beta-subunit catalytic activities.

Benzimidazoles↗
PubMed1993

Amino acid substitutions at position 47 of human beta 1 beta 1 and beta 2 beta 2 alcohol dehydrogenases affect hydride transfer and coenzyme dissociation rate constants.

Human liver alcohol dehydrogenase isoenzymes beta 1 beta 1 and beta 2 beta 2, in which position 47 in the coenzyme binding domain is an arginine or histidine, respectively, differ remarkably in steady-state kinetics. To understand which catalytic steps affect these kinetics, apparent coenzyme dissociation and association rate constants, and apparent 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) hydride transfer rate constants were obtained with stopped-flow kinetics. Enzymes containing site-specific mutations of Arg-47 in beta 1 beta 1 (beta 47R) to His (beta 2 beta 2 or beta 47H), Lys (beta 47K), or Gln (beta 47Q) were studied. Apparent coenzyme dissociation rate constants are greatly affected by substitutions at position 47, in which mutant enzymes with a weak base or a neutral residue at this position (beta 47H and beta 47Q) exhibit faster rate constants than beta 47R and beta 47K. Substitutions at position 47 have less effect on apparent coenzyme association rate constants. The kinetics of NADH association for beta 47H and beta 47Q are consistent with a two-step mechanism in which the bimolecular binding step is coupled to a unimolecular process. These findings indicate that the greater role of position 47 in coenzyme dissociation may occur after a coenzyme-induced isomerization. Substitutions at position 47 also strongly influence apparent DACA hydride transfer rate constants; hydride transfer is faster with mutant enzymes containing weak bases like histidine at this position. Steady-state kinetics, however, reveal that the rate-limiting step of both beta 47R and beta 47H for acetaldehyde reduction and for ethanol oxidation is coenzyme product dissociation. Thus, the different activities of beta 1 beta 1 and beta 2 beta 2 for ethanol oxidation and acetaldehyde reduction are caused primarily by different coenzyme dissociation rates.

Alcohol Dehydrogenase↗