Kinetics of a glycine for Arg-47 human alcohol dehydrogenase mutant can be explained by Lys-228 recruitment into the pyrophosphate binding site.
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Alcohol Dehydrogenase↗
Biomedical subjects
M F Dunn
Publications and source records attributed to M F Dunn.
Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.
Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction. However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., Brzović, P., Drewe, W.F., Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed" structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open to the closed form.
Allosteric Regulation↗
The T to R transition in the copper(II)-substituted insulin hexamer. Anion complexes of the R-state species exhibiting type 1 and type 2 spectral characteristics.
The R-state conformation of the Cu(II)-substituted insulin hexamer has been identified, and a number of its derivatives have been studied via 1H NMR, ESR, and UV-visible spectroscopy. This work establishes that the Cu(II)-substituted insulin hexamer undergoes an analogous T to R conformational transition in solution that has been identified previously for Zn(II)- and Co(II)-insulin hexamers [Roy, M., Brader, M.L., Lee, R. W.-K., Kaarsholm, N.C., Hansen, J., & Dunn, M.F. (1989) J. Biol. Chem. 264, 19081-19085]. The data indicate that each Cu(II) center of the R-state Cu(II)-insulin hexamer possesses a coordination site that is accessible to anions from solution. Both phenol and anionic ligands that coordinate to the Cu(II) ions are required to generate the necessary heterotropic interactions that stabilize the R-state structure. With phenylmethylthiolate (PMT), a Cu(II)-R6 adduct that displays the spectral features of blue (type 1) copper proteins is obtained. This complex is proposed to embody a pseudotetrahedral CuIIN3S(PMT) chromophore, in which N is HisB10 (imidazolyl). The remaining ligands examined gave rise to Cu(II)-R6 adducts that possessed the spectral characteristics of normal (type 2) Cu(II) proteins. Under reducing conditions, Cu(I)-T6 and Cu(I)-R6 hexamers have been identified.
Copper↗
Studies of the association and conformational properties of metal-free insulin in alkaline sodium chloride solutions by one- and two-dimensional 1H NMR.
One- and two-dimensional 1H NMR spectroscopy have been employed to probe the association and subsequent conformational changes of metal-free insulin in sodium chloride solution at pH 9 and 9.4. These studies establish that the proton resonances of His(B5) and His(B10) are useful signatures of aggregation and conformation. Changes in chemical shifts and areas of resonances due to the C2 protons of His(B10) and His(B5) and transfer of magnetization experiments served to identify the association as the assembly of tetramer from dimers under our experimental conditions (pH 9.4, [insulin] greater than 1 mM, [NaCl] = 0.1 M). Sodium chloride also alters the equilibrium distribution of species in favor of a tetrameric species. The association equilibrium constant was estimated from area measurements to be approximately 5 x 10(3) M-1 at pH 9.4, 26 +/- 0.1 degrees C, and 0.1 M sodium chloride. Under conditions of 0.1 M sodium chloride concentration, nuclear Overhauser effect experiments in the one- and two-dimensional modes revealed an operative nuclear Overhauser effect between the His(B5) C2 protons and the 2,6 ring protons of a Tyr residue provisionally assigned as Tyr(B16). We conclude that this interaction is a diagnostic signature of a conformational transition whereupon an extended chain from residues B1 to B9 (T-state) is transformed into an alpha-helix (R-state) thus bringing the rings of His(B5) and Tyr(B16) from adjacent subunits across the monomer-monomer interface into van der Waals contact. This conformational flexibility is an added consideration to the discussion of the relevant structure of insulin for receptor binding.
Animals↗
Allosteric interactions coordinate catalytic activity between successive metabolic enzymes in the tryptophan synthase bienzyme complex.
Tryptophan synthase from enteric bacteria is an alpha 2 beta 2 bienzyme complex that catalyzes the final two reactions in the biosynthesis of L-tryptophan (L-Trp) from 3-indole-D-glycerol 3'-phosphate (IGP) and L-serine (L-Ser). The bienzyme complex exhibits reciprocal ligand-mediated allosteric interactions between the heterologous subunits [Houben, K., & Dunn, M. F. (1990) Biochemistry 29, 2421-2429], but the relationship between allostery and catalysis had not been completely defined. We have utilized rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy to study the relationship between allostery and catalysis in the alpha beta-reaction catalyzed by the bienzyme complex from Salmonella typhimurium. The pre-steady-state spectral changes that occur when L-Ser and IGP are mixed simultaneously with the alpha 2 beta 2 complex show that IGP binding to the alpha-site accelerates the formation of alpha-aminoacrylate [E(A-A)] from L-Ser at the beta-site. Through the use of L-Ser analogues, we show herein that the formation of the E(A-A) intermediate is the chemical signal which triggers the conformational transition that activates the alpha-subunit. beta-subunit ligands, such as L-Trp, that react to form covalent intermediates at the beta-site, but are incapable of E(A-A) formation, do not stimulate the activity of the alpha-subunit. Titration experiments show that the affinity of G3P and GP at the alpha-site is dependent upon the nature of the chemical intermediate present at the beta-active site.(ABSTRACT TRUNCATED AT 250 WORDS)
Alanine↗
Spectroscopic evidence for an intermediate in the T6 to R6 allosteric transition of the Co(II)-substituted insulin hexamer.
The phenol-induced conformational transition in the insulin hexamer is known to involve a large change in structure wherein residues 1-8 of the insulin B-chain are transformed from an extended coil (T-state) to a helix (R-state). This change in protein conformation both exposes a cryptic protein pocket on each subunit to which phenol binds and forces the HisB10 zinc sites to undergo a change in coordination geometry from octahedral to tetrahedral [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 593-596]. Substitution of Co(II) for Zn(II) at the HisB10 sites introduces a sensitive chromophoric probe of the structural and chemical events that occur during this allosteric transition [Roy, M., Brader, M. L., Lee, R. W.-K., Kaarsholm, N. C., Hansen, J. F., & Dunn, M. F. (1989) J. Biol. Chem. 264, 19081-19085]. In this study, using rapid-scannig stopped-flow (RSSF) UV-visible spectroscopic studies, we demonstrate that a transient chemical intermediate is formed during the phenol-induced conversion of Co(II)-substituted hexamer from the T-state to the R-state. Decomposition of the RSSF spectra gave a spectrum for the intermediate with d-d transitions consistent with the assignment of the intermediate as either a distorted tetrahedral or a 5-coordinate Co(II) species. Possible structures for the intermediate and the implications of these findings to the allosteric mechanism are considered.
Allosteric Site↗
Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the beta-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium.
In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.
Aspartic Acid↗
Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes.
Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.
Arginine↗
Characterization of the R-state insulin hexamer and its derivatives. The hexamer is stabilized by heterotropic ligand binding interactions.
1H NMR and UV-visible electronic absorption studies have been performed to investigate the effects of anions and cyclic organic molecules on the interconversion of the T- and R-conformational states (Kaarsholm et al., 1989) of hexameric M (II)-substituted insulin in solution (M = Zn or Co.). Two ligand binding processes that stabilize the R-state conformation of the M(II)-substituted insulin hexamer [M(II)-R6] have been distinguished: (i) The binding of neutral organic molecules to the six, crystallographically identified, protein pockets in the Zn(II)-R6 insulin hexamer (Derewenda et al. 1989) generate homotropic site-site interactions that stabilize the R-state. Cyclohexanol, phenol, 4-nitrophenol, and 4-hydroxymethylbenzoate are shown to bind at these sites. (ii) The coordination of singly charged anions that are able to gain access to the two HisB10 coordinated metal ions of the M(II)-R6 hexamer stabilizes the R-state. Adducts of the M(II)-R6 hexamer are formed, thereby, in which the solvent-accessible fourth coordination position of the M(II) ion is replaced by a competing anion. Binding to these two classes of sites introduces strong heterotropic interactions that stabilize the R-state. UV-visible spectral data and apparent affinity constants for the adducts formed by the Co(II)-R6 hexamer with a wide range of anionic ligands are presented. The Co(II)-R6 adducts have a strong preference for the formation of pseudotetrahedral Co(II) centers. The HCO3- and pyridine-2-thiolate ions form Co(II)-R6 adducts that are proposed to possess pentacoordinate Co(II) geometries. The relevance of the Co(II)-R6 complexes to carbonic anhydrase catalysis and zinc enzyme model systems is discussed.
Biopolymers↗
X-ray structure of an unusual Ca2+ site and the roles of Zn2+ and Ca2+ in the assembly, stability, and storage of the insulin hexamer.
Metal ion binding to the insulin hexamer has been investigated by crystallographic analysis. Cadmium, lead, and metal-free hexamers have been refined to R values of 0.181, 0.172, and 0.172, against data of 1.9-, 2.5-, and 2.5-A resolution, respectively. These structures have been compared with each other and with the isomorphous two-zinc insulin. The structure of the metal-free hexamer shows that the His(B10) imidazole rings are arranged in a preformed site that binds a water molecule and is poised for Zn2+ coordination. The structure of the cadmium derivative shows that the binding of Cd2+ at the center of the hexamer is unusual. There are three symmetry-related sites located within 2.7 A of each other, and this position is evidently one-third occupied. It is also shown that the coordinating B13 glutamate side chains of this derivative have two partially occupied conformations. One of these conformations is two-thirds occupied and is very similar to that seen in two-zinc insulin. The other, one-third-occupied conformation, is seen to coordinate the one-third-occupied metal ion. The binding of Ca2+ to insulin is assumed to be essentially identical with that of Cd2+. Thus, we conclude that the Ca2+ binding site in the insulin hexamer is unlike that of any other known calcium binding protein. The crystal structures reported herein explain how binding of metal ions stabilizes the insulin hexamer. The role of metal ions in hexamer assembly and dissociation is discussed.
Animals↗
Insulin hexamers: new conformations and applications.
Recent studies on the structural and chemical properties of insulin have shown that the insulin hexamer is an allosteric protein capable of adopting three distinct conformations, designated T6, T3R3 and R6. Although the physiological consequences of this allostery are not established, new applications for the insulin hexamer as a model system for the study of allostery and for the study of zinc enzymes and copper proteins are emerging.
Insulin↗
The tryptophan synthase bienzyme complex transfers indole between the alpha- and beta-sites via a 25-30 A long tunnel.
The bacterial tryptophan synthase bienzyme complexes (with subunit composition alpha 2 beta 2) catalyze the last two steps in the biosynthesis of L-tryptophan. For L-tryptophan synthesis, indole, the common metabolite, must be transferred by some mechanism from the alpha-catalytic site to the beta-catalytic site. The X-ray structure of the Salmonella typhimurium tryptophan synthase shows the catalytic sites of each alpha-beta subunit pair are connected by a 25-30 A long tunnel [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Since the S. typhimurium and Escherichia coli enzymes have nearly identical sequences, the E. coli enzyme must have a similar tunnel. Herein, rapid kinetic studies in combination with chemical probes that signal the bond formation step between indole (or nucleophilic indole analogues) and the alpha-aminoacrylate Schiff base intermediate, E(A-A), bound to the beta-site are used to investigate tunnel function in the E. coli enzyme. If the tunnel is the physical conduit for the transfer of indole from the alpha-site to the beta-site, then ligands that block the tunnel should also inhibit the rate at which indole and indole analogues from external solution react with E(A-A). We have found that when D,L-alpha-glycerol 3-phosphate (GP) is bound to the alpha-site, the rate of reaction of indole and nucleophilic indole analogues with E(A-A) is strongly inhibited. These compounds appear to gain access to the beta-site via the alpha-site and the tunnel, and this access is blocked by the binding of GP to the alpha-site. However, when small nucleophiles such as hydroxylamine, hydrazine, or N-methylhydroxylamine are substituted for indole, the rate of quinonoid formation is only slightly affected by the binding of GP. Furthermore, the reactions of L-serine and L-tryptophan with alpha 2 beta 2 show only small rate effects due to the binding of GP. From these experiments, we draw the following conclusions: (1) L-Serine and L-tryptophan gain access to the beta-site of alpha 2 beta 2 directly from solution. (2) The small effects of GP on the rates of the L-serine and L-tryptophan reactions are due to GP-mediated allosteric interactions between the alpha- and beta-sites.(ABSTRACT TRUNCATED AT 400 WORDS)
Allosteric Regulation↗
Mechanism of binding of substrate analogues to tryptophan indole-lyase: studies using rapid-scanning and single-wavelength stopped-flow spectrophotometry.
We have examined the binding of oxindolyl-L-alanine, (3R)-2,3-dihydro-L-tryptophan, L-homophenylalanine, and N1-methyl-L-tryptophan to tryptophan indole-lyase (tryptophanase) from Escherichia coli by using rapid-scanning and single-wavelength stopped-flow kinetic techniques. Rate constants for the reactions were determined by fitting the concentration dependencies of relaxations to either linear (pseudo-first-order) or hyperbolic (rapid second-order followed by slow first-order) equations. The reaction with oxindolyl-L-alanine forms a quinonoid intermediate that exhibits a strong peak at 506 nm. This species is formed more rapidly than with the other analogues (84.5 s-1) and is reprotonated very slowly (0.2 s-1). Reaction with L-homophenylalanine also forms a quinonoid intermediate with a strong peak at 508 nm, but the rate constant for its formation is slower (6.9 s-1). The reaction with L-homophenylalanine exhibits a transient intermediate absorbing at about 340 nm that decays at the same rate as the quinonoid peak forms and that may be a gem-diamine. Tryptophan indole-lyase reacts with (3R)-2,3-dihydro-L-tryptophan much more slowly to form a moderately intense quinonoid peak at 510 nm, and a transient intermediate absorbing at about 350 nm is also formed. The species formed in the reaction of N1-methyl-L-tryptophan exhibits a peak at 425 nm and a very weak quinonoid absorption peak at 506 nm, which is formed at less than 4 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Alanine↗
The R-state proinsulin and insulin hexamers mimic the carbonic anhydrase active site.
The cobalt(II)-substituted proinsulin and insulin hexamers have been studied in solution via electronic absorption spectroscopy. Hexameric proinsulin is shown to undergo the phenol-induced T6 to R6 conformational transition in a manner analogous to that previously established for insulin. In the absence of coordinating anions, the coordination spheres of the Co(II) ions in the proinsulin and insulin R6 hexamers comprise identical pseudotetrahedral arrangements of 3 histidine residues and 1 hydroxide ion. At alkaline pH, the visible absorption spectrum of the phenol-induced R6 Co(II) center is strikingly similar to the distinctive spectrum of the alkaline form of Co(II)-carbonic anhydrase. Exogenous ligands may coordinate to the Co(II) ions of the R6 proinsulin and insulin hexamers via replacement of the hydroxide ion, forming pseudotetrahedral adducts possessing characteristic spectra. The binding affinity of such ligands is shown to be strongly pH-dependent. The data presented establish that, although the Co(II)-substituted proinsulin and insulin R6 hexamers lack enzyme-like activity, these species duplicate spectrochemical characteristics of the Co(II)-carbonic anhydrase active site that are believed to be important signatures of carbonic anhydrase catalytic function.
Animals↗
Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin.
The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.
Amino Acid Sequence↗
1H NMR spectrum of the native human insulin monomer. Evidence for conformational differences between the monomer and aggregated forms.
The effects of high dilution on the 1H Fourier transform NMR spectrum of native human insulin at pH* 8.0 and 9.3 have been examined at 500 MHz resolution. The dependence of the spectrum on concentration and comparison with the spectrum of a biologically highly potent monomeric insulin mutant (SerB9----Asp) establish that at 36 microM (pH* 9.3) or 18 microM (pH* 8) and no added buffer or salts, human insulin is monomeric. Under these conditions of dilution, ionic strength, and pH*, human insulin and the SerB9----Asp mutant exhibit nearly identical 1H NMR spectra. At higher concentrations (i.e. greater than 36 microM to 0.91 mM), native human insulin dimerizes, and this aggregation causes a change in insulin conformation. Although there are many changes in the spectrum, the TyrB26 ring H3,5 proton signals located at 6.63 ppm and the methyl signal located at 0.105 ppm (characteristics of monomeric insulin) are particularly distinct signatures of the conformation change that accompanies dimerization. Magnetization transfer experiments show that the 0.105 ppm methyl signal shifts downfield to a new position at 0.45 ppm. We conclude that the 0.105 ppm methyl signal is due to a conformation in which a Leu methyl group is centered over and in van der Waals contact with the ring of an aromatic side chain. Dimerization causes a conformation change that alters this interaction, thereby causing the downfield shift. Nuclear Overhauser studies indicate that the methyl group involved is located within a cluster of aromatic side chains and that the closest ring-methyl group interaction is with the ring of PheB24.
Aspartic Acid↗
Allosteric effects acting over a distance of 20-25 A in the Escherichia coli tryptophan synthase bienzyme complex increase ligand affinity and cause redistribution of covalent intermediates.
The reactions of L-histidine (L-His) and L-tryptophan (L-Trp) with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase are introduced as probes both of beta-subunit catalysis and of ligand-mediated alpha-beta allosteric interactions. Binding of DL-alpha-glycerol 3-phosphate (GP), an analogue of 3-indole-D-glycerol 3'-phosphate (IGP), to the alpha-catalytic site increases the affinity of alpha 2 beta 2 for L-His 4.5-fold and the affinity for L-Trp 17-fold and brings about a redistribution of beta-bound intermediates that favors the quinonoids derived from each amino acid. Inorganic phosphate (Pi) (presumably via binding to the alpha-catalytic site) influences the distribution of L-His intermediates as does GP. Previous binding studies [Heyn, M. P., & Weischet, W. O. (1975) Biochemistry 14, 2962-2968] indicate that when the phosphoryl group subsite of the alpha-catalytic site is occupied by GP or Pi, a high-affinity indole subsite is induced at the alpha-catalytic site. Interaction of benzimidazole (BZ), an analogue of indole, with this site also shifts the distribution of beta-bound L-His intermediates in favor of the L-His quinonoid. In the absence of Pi or GP, BZ interacts primarily at the beta-catalytic site and competes with L-His for the beta-subunit indole subsite. Since L-His and GP (or Pi) are substrate analogues and L-Trp is the physiological product, these allosteric effects likely take place with the natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
Allosteric Regulation↗
Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions.
Cystathionine gamma-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a gamma-replacement reaction. In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, gamma-elimination reaction to form succinate, alpha-ketobutyrate, and ammonia. Since elimination of the gamma-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate. Previously, this partitioning intermediate has been assigned to a highly conjugated alpha-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the gamma-replacement and gamma-elimination reactions of cystathionine gamma-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The gamma-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region. A 485-nm species subsequently accumulates in a much slower relaxation. The gamma-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (approximately 3 ms). This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region. Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine gamma-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species. In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allylglycine undergoes a gamma-replacement reaction to form a new amino acid, gamma-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.
Alanine↗