Metabolite channelling in tryptophan synthase.
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Indoles↗
Biomedical subjects
A M Kayastha
Publications and source records attributed to A M Kayastha.
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↗
Amperometric enzyme sensor for glucose based on graphite paste-modified electrodes.
Amperometric enzyme electrode for glucose is described based on the incorporation of glucose oxidase (GOD) into graphite paste modified with tetracyanoquinodimethane (TCNQ). The incorporated enzyme exhibits high activity and long-term stability over the earlier TCNQ-based glucose sensor (1). The sensor provides a linear response to glucose over a wide concentration range. The response time of the sensor is 15-50 sec, and the detection limit is 0.5 mM. Stable response to the substrate was obtained during a period of 35 d. Application of the sensor in the plasma analysis is reported.
Biosensing Techniques↗
Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.
Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Mechanism of mutual activation of the tryptophan synthase alpha and beta subunits. Analysis of the reaction specificity and substrate-induced inactivation of active site and tunnel mutants of the beta subunit.
The origin of reaction and substrate specificity and the control of activity by protein-protein interaction are investigated using the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. We have compared some spectroscopic and kinetic properties of the wild type beta subunit and five mutant forms of the beta subunit that have altered catalytic properties. These mutant enzymes, which were engineered by site-directed mutagenesis, have single amino acid replacements in either the active site or in the wall of a tunnel that extends from the active site of the alpha subunit to the active site of the beta subunit in the alpha 2 beta 2 complex. We find that the mutant alpha 2 beta 2 complexes have altered reaction and substrate specificity in beta-elimination and beta-replacement reactions with L-serine and with beta-chloro-L-alanine. Moreover, the mutant enzymes, unlike the wild type alpha 2 beta 2 complex, undergo irreversible substrate-induced inactivation. The mechanism of inactivation appears to be analogous to that first demonstrated by Metzler's group for inhibition of two other pyridoxal phosphate enzymes. Alkaline treatment of the inactivated enzyme yields apoenzyme and a previously described pyridoxal phosphate derivative. We demonstrate for the first time that enzymatic activity can be recovered by addition of pyridoxal phosphate following alkaline treatment. We conclude that the wild type and mutant alpha 2 beta 2 complexes differ in the way they process the amino acrylate intermediate. We suggest that the wild type beta subunit undergoes a conformational change upon association with the alpha subunit that alters the reaction specificity and that the mutant beta subunits do not undergo the same conformational change upon subunit association.
Base Sequence↗
Site-directed mutagenesis of the beta subunit of tryptophan synthase from Salmonella typhimurium. Role of active site glutamic acid 350.
To investigate the functional role of glutamic acid 350 in the active site of the beta subunit of tryptophan synthase from Salmonella typhimurium, we have replaced this residue by glutamine or alanine by use of site-directed mutagenesis. The mutant alpha 2 beta 2 complexes were expressed, purified, crystallized, and characterized by spectroscopic and kinetic studies with several substrates. We find large alterations in the substrate and reaction specificity of each mutant form of the alpha 2 beta 2 complex. Since the two mutant enzymes are virtually inactive in reactions with L-serine but are active in reactions with beta-chloro-L-alanine, glutamic acid 350 may facilitate the beta-elimination of the weak hydroxyl leaving group of L-serine. The mutant alpha 2 beta 2 complexes are more active than the wild type enzyme in the beta-elimination reaction with beta-chloro-L-alanine. These enzymes are irreversibly inactivated by beta-chloro-L-alanine, whereas the wild type enzyme is not. These altered properties may result from a change in the conformation of the active site, from a change in the orientation of the coenzyme relative to active site residues, or from a change in the solvent accessibility of the active site. The alteration in the active site may enhance the release of amino acrylate from the Schiff base intermediate by hydrolysis or by transamination.
Amino Acids↗
A colorimetric assay for a pyridoxal phosphate-dependent beta-replacement reaction with L-cysteine: application to studies of wild-type and mutant tryptophan synthase alpha 2 beta 2 complexes.
We present an improved and simple direct assay for formation of inorganic sulfide from L-cysteine in a beta-replacement reaction catalyzed by tryptophan synthase. This method provides a useful enzymatic assay for pyridoxal phosphate-dependent beta-replacement reactions in which the amino acid substrate is L-cysteine and the cosubstrate is 2-mercaptoethanol. The assay should be applicable to similar reactions with L-cysteine and other cosubstrates. The method has several advantages over other methods which have been used to assay similar beta-replacement reactions. The assay is highly reproducible and sensitive and is conveniently carried out in disposable 1.5-ml centrifuge tubes. The color remains stable for several hours. The thiol compounds L-cysteine and 2-mercaptoethanol do not interfere at the concentrations used. The method has useful applications to studies of the rates and reaction specificities of several other pyridoxal phosphate enzymes which catalyze beta-replacement reactions. We demonstrate the use of the method to study the effects of site-directed mutagenesis on the reaction specificity and mechanism of the tryptophan synthase alpha 2 beta 2 complex.
Colorimetry↗
Circular dichroism studies of the coenzyme environment in the active sites of mutant forms of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex.
The circular dichroism has been used to evaluate the effect of mutation on the environment of the pyridoxal phosphate coenzyme in the active site of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Seven mutant forms of the alpha 2 beta 2-complex with single amino acid replacements at residues 87, 109, 188, 306, and 350 of the beta-subunit have been prepared by site-directed mutagenesis, purified to homogeneity, and characterized by absorption and circular dichroism spectroscopy. Since the wild type and mutant alpha 2 beta 2 complexes all exhibit positive circular dichroism in the coenzyme absorption band, pyridoxal phosphate must bind asymmetrically in the active site of these enzymes. However, the coenzyme may have an altered orientation or active site environment in five of the mutant enzymes that display less intense ellipticity bands. The mutant enzyme in which lysine 87 is replaced by threonine has very weak ellipticity at 400 nm. Since lysine 87 forms a Schiff base with pyridoxal phosphate in the wild type enzyme, our results demonstrate the importance of the Schiff base linkage for rigid or asymmetric binding. Although the mutant enzymes display spectra in the presence of L-serine that differ from that of the wild type enzyme, addition of alpha-glycerol 3-phosphate converts the spectra of two of the mutant enzymes to that of the wild type enzyme. We conclude that this alpha-subunit ligand may produce a conformational change in the alpha-subunit that is transmitted to the mutant beta-subunits and partially corrects conformational alterations in the mutant enzymes.
Base Sequence↗