Edith Wilson Miles

[41] Modification of histidyl residues in proteins by diethylpyrocarbonate

Publisher Summary Diethylpyrocarbonate has been shown to react specifically or stoichiometrically with a single histidyl residue in certain proteins. In other cases, the modification of activity has been correlated with the modification of one or more histidyl residues despite the possible modification of other residues; this correlation is facilitated by the fact that hydroxylamine removes the carbethoxy group from modified histidyl residues and tyrosyl residues, but not that of modified lysyl or sulfhydryl residues. Several enzymes have been shown to be inactivated by the modification of a residue other than a histidyl residue. Thus, although diethylpyrocarbonate does not always react specifically with histidyl residues in proteins, it is more selective than other acylating agents and can give useful information about the role of histidyl residues in many proteins. This chapter describes the way optimal conditions for reaction should be determined and the way possible side reaction should be examined.

The 8 protein of Escherichia coli tryptophan synthetase II. New β-elimination and β-replacement reactions

The B protein of Escherichia coli tryptophan synthetase is reported to catalyze β-elimination reactions with S-methyl-L-cysteine, O-methyl-L-serine, and β-chloro-L-alanine to produce pyruvate and β-replacement reactions with the same substrates plus indole to produce L-tryptophan. The mechanism of this enzyme is comparable to that of tryptophanase of E. coli , but the two enzymes have some important differences in reaction specificity.

Enzymatic synthesis of Se-substituted L-selenocysteine with tryptophan synthase

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A new type of pyridoxal-p enzyme catalyzed reaction: The conversion of β, γ-unsaturated amino acids to saturated α-keto acids by tryptophan synthase

2-Amino-3-butenoic acid (vinyl glycine) and trans -L-2-amino-4-methoxy-3-butenoic acid are converted to saturated α-keto acids: α-ketobutyrate and α-keto-4-methoxybutyrate, respectively, by tryptophan synthase of Escherichia coli . A sequence of pyridoxal-P Schiff base intermediates is proposed for these reactions. The discovery of these pyridoxal-P dependent reactions of β,γ-unsaturated amino acids lends weight to previous proposals by others that pyridoxal-P Schiff base derivatives of β,γ-unsaturated amino acids are intermediates in γ-elimination reactions and in γ,β-isomerization reactions.

Detection of open and closed conformations of tryptophan synthase by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance of bound 1-15N-L-tryptophan.

1-15N-l-Tryptophan (1-15N-l-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with l-serine, catalyzed by tryptophan synthase. 1-15N-l-Trp was complexed with wild-type tryptophan synthase and β-subunit mutants, βK87T, βD305A, and βE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium α-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1-15N-l-Trp bound to the β-active site. No 15N-HSQC signal was detected for 1-15N-l-Trp in 10 mm triethanolamine hydrochloride buffer at pH 8. 1-15N-l-Trp in the presence of wild-type tryptophan synthase in the absence or presence of 50 mm sodium chloride showed a cross peak at 10.25 ppm on the 1H axis and 129 ppm on the 15N axis as a result of reduced solvent exchange for the bound 1-15N-l-Trp, consistent with formation of a closed conformation of the active site. The addition of disodium α-glycerophosphate produced a signal twice as intense, suggesting that the equilibrium favors the closed conformation. 15N-HSQC NMR spectra of βK87T and βE109D mutant Trp synthase with 1-15N-l-Trp showed a similar cross peak either in the presence or absence of disodium α-glycerophosphate, indicating the preference for a closed conformation for these mutant proteins. In contrast, the βD305A Trp synthase mutant only showed a 15N-HSQC signal in the presence of disodium α-glycerophosphate. Thus, this mutant Trp synthase favored an open conformation in the absence of disodium α-glycerophosphate but was able to form a closed conformation in the presence of disodium α-glycerophosphate. Our results demonstrate that the 15N-HSQC NMR spectra of 1-15N-l-Trp bound to Trp synthase can be used to determine the conformational state of mutant forms in solution rapidly. In contrast, UV-visible spectra of wild-type and mutant Trp synthase in the presence of l-Trp with NaCl and/or disodium α-glycerophosphate are more difficult to interpret in terms of altered conformational equilibria.

Overexpression and purification of the separate tryptophan synthase α and β subunits from Salmonella typhimurium

To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.

Comparison of denaturation of tryptophan synthase α-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase alpha subunit have been compared by circular dichroism measurements. The three alpha subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (alpha-2 domain) and the N-terminal domain comes from S. typhimurium (alpha-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 degrees C showed that the alpha-2 domain of S. typhimurium was more stable than the alpha-2 domain of E. coli, but the alpha-1 domain of S. typhimurium was less stable than the alpha-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 degrees C and coincided with that obtained from guanidine hydrochloride-induced denaturation.

Mechanism of Activation of the Tryptophan Synthase α2β2 Complex SOLVENT EFFECTS OF THE CO-SUBSTRATE β-MERCAPTOETHANOL

To characterize the conformational transitions that lead to activation of catalysis by the tryptophan synthase α2β2 complex, we have determined the solvent effects of a co-substrate, β-mercaptoethanol, and of a model nonsubstrate, ethanol, on the catalytic and spectroscopic properties of the enzyme. Our results show that ethanol and β-mercaptoethanol both alter the equilibrium distribution of pyridoxal 5′-phosphate intermediates formed in the reactions of L-serine at the β site in the α2β2 complex. Addition of increasing concentrations of ethanol increases the proportion of the external aldimine of L-serine and decreases the proportion of the external aldimine of aminoacrylate. Low concentrations of the co-substrate β-mercaptoethanol (Kd = ∼13 mM) decrease the proportion of the external aldimine of aminoacrylate and induce formation of the quinonoid of S-hydroxyethyl-L-cysteine. Higher concentrations of β-mercaptoethanol decrease the concentration of the quinonoid intermediate and increase the proportion of the external aldimine of L-serine. Data analysis shows that β-mercaptoethanol and ethanol both interact or bind preferentially with the conformer of the enzyme that predominates when the aldimine of L-serine is formed and shift the equilibrium in favor of this conformer. We propose that a nonpolar region of the β subunit, possibly the hydrophobic indole tunnel, becomes less exposed to solvent in the conformational transition that activates the α2β2 complex.

Pressure and Temperature Jump Relaxation Kinetics of the Conformational Change in Salmonella typhimurium Tryptophan Synthase l-Serine Complex: Large Activation Compressibility and Heat Capacity Changes Demonstrate the Contribution of Solvation

Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex. The intense fluorescence emission of the Tryptophan synthase L-Ser external aldimine complex at 495 nm, with 420 nm excitation, provides a probe of the conformational state of Trp synthase. P-jump measurements allowed the determination of rate constants for the reactions in the presence of Na(+), Na(+) with benzimidazole (BZI), and NH4(+). The data require a compressibility term, beta(o)(double dagger), to obtain good fits, especially for the NH4(+) and BZI/Na(+) data. The compressibility changes are consistent with changes in solvation in the transition state. The transition state for the relaxation is more similar in volume to the closed aminoacrylate complex in the presence of Na(+), while it is more similar to the open external aldimine in the presence of NH4(+). Differences between the relaxations for positive and negative P-jumps may arise from changing relative populations of microstates with pressure. T-jump experiments of the Na(+) form of the tryptophan synthase-L-Ser complex show large changes in rate and amplitude over the temperature range from 7 to 45 degrees C. The Arrhenius plots show strong curvature, and hence require a heat capacity term, DeltaC(p)(double dagger), to obtain good fits. The values of DeltaC(p)(double dagger) are very large and negative (-3.6 to -4.4 kJ mol(-1) K(-1)). These changes are also consistent with large changes in solvation in the transition state for interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.

Location of the reactive sulfhydryl residues in the primary sequence of the β2 subunit of tryptophan synthase of Escherichiacoli

Abstract Two of the 5 sulfhydryl residues of the β2 subunit of tryptophan synthase have previously been shown to react with N-ethylmaleimide and to have active site roles. We now show that the single sulfhydryl which reacts with N-ethylmaleimide in the presence of pyridoxal phosphate is cysteine-170. The essential sulfhydryl which reacts with N-ethylmaleimide or with 2-nitro-5-thiocyanobenzoic acid after removal of pyridoxal phosphate is cysteine-230. The affinity reagent, bromoacetylpyridoxamine phosphate, reacts variably with cysteine-62 or with cysteine-230.