Detection and identification of transient intermediates in the reactions of tryptophan synthase with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Evidence for a tetrahedral (gem-diamine) intermediate

The reactions of 2,3-dihydro-L-tryptophan (DHT) and oxindolyl-L-alanine (OXA) with tryptophan synthase have been investigated by rapid-scanning stopped-flow (RSSF) spectroscopy and by the concentration dependence of rates measured by single-wavelength stopped-flow (SWSF) spectroscopy. The RSSF spectral changes for DHT and OXA show the disappearance of the internal aldimine (lambda max 412 nm), the formation and decay of intermediates absorbing less than or equal to 340 nm, and the appearance of the quinonoid (lambda max 492 and 480 nm, respectively). Rate constants determined by SWSF were either well resolved (i.e., k1[DHT], k-1 greater than k2, k-2 greater than k3, k-3) or indicative of a tightly coupled system (i.e., k1[OXA], k-1 greater than or equal to k2, k-2 greater than k3, k-3). The RSSF spectral changes and SWSF kinetic studies together with computer simulations of the kinetic time courses are consistent with a mechanism that includes formation of a bleached species. Detection of these shorter wavelength species in the reactions of OXA and DHT indicates that substrate analogues with tetrahedral geometry at C-3 induce new protein-substrate interactions that result in the accumulation of species not previously detected in the tryptophan synthase system. The bleached species with lambda max less than or equal to 340 nm are proposed as the gem-diamine intermediates.     

Differential inhibition of tryptophan synthase and of tryptophanase by the two diastereoisomers of 2,3-dihydro-L-tryptophan. Implications for the stereochemistry of the reaction intermediates

Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan, which are analogs of a proposed reaction intermediate, are potent competitive inhibitors of both tryptophanase and the alpha 2 beta 2 complex of tryptophan synthase (Phillips, R. S., Miles, E. W., and Cohen, L. A. (1984) Biochemistry 23, 6228-6234). Since these inhibitors can exist in two diastereoisomeric forms, which we expected to differ in inhibitory potency, we have separated the diastereoisomers of 2,3-dihydro-L-tryptophan by preparative high performance liquid chromatography. These diastereoisomers were designated "A" and "B" in order of elution from the high performance liquid chromatography column. Diastereoisomer B is a potent competitive inhibitor of the alpha 2 beta 2 complex of tryptophan synthase with KI = 6 microM at pH 7.8 and 25 degrees C. In contrast, diastereoisomer A is a weak competitive inhibitor, with KI = 940 microM under these conditions. With tryptophanase, the situation is reversed; diastereoisomer A is a potent slow-binding competitive inhibitor of tryptophanase with KI = 2 microM at pH 8.0 and 25 degrees C, while diastereoisomer B is much weaker with KI = 1600 microM under these conditions. These results not only provide additional support for the proposal that the indolenine tautomer of tryptophan is an intermediate in the reactions catalyzed by both enzymes but also suggest that these enzymes catalyze their respective reactions via enantiomeric indolenine intermediates.     

Neurospora tryptophan synthase. Characterization of the pyridoxal phosphate binding site

Tryptophan synthase, which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein that requires pyridoxal phosphate for two of its three distinct enzyme activities. Tryptophan synthase from Neurospora crassa, a homodimer of two 75-kDa subunits, was shown to bind 1 mol of pyridoxal phosphate/mol of subunit with a calculated dissociation constant for pyridoxal phosphate of 1.1 microM. The spectral properties of the holoenzyme, apoenzyme, and reconstituted holoenzyme were characterized and compared to those previously established for the heterotetrameric (alpha 2 beta 2) enzyme from Escherichia coli. The Schiff base formed between pyridoxal phosphate and the enzyme was readily reduced by sodium borohydride, but not sodium cyanoborohydride. The active site residue that binds pyridoxal phosphate, labeled by reduction of the Schiff base with tritium-labeled sodium borohydride, was determined to be lysine by high performance liquid chromatography analysis of the protein hydrolysate. A 5400-dalton peptide containing the reduced pyridoxal phosphate moiety was generated by cyanogen bromide treatment, purified and sequenced. The sequence is 85% homologous with the corresponding sequence obtained for yeast tryptophan synthase (Zalkin, H., and Yanofsky, C. (1982) J. Biol. Chem. 257, 1491-1500); the lysine derivatized by pyridoxal phosphate is located at the same relative position as that in the yeast and E. coli enzymes.     

Reaction mechanism of pyridoxal 5'-phosphate synthase. Detection of an enzyme-bound chromophoric intermediate

Vitamin B6 is an essential metabolite in all organisms. De novo synthesis of the vitamin can occur through either of two mutually exclusive pathways referred to as deoxyxylulose 5-phosphate-dependent and deoxyxylulose 5-phosphate-independent. The latter pathway has only recently been discovered and is distinguished by the presence of two genes, Pdx1 and Pdx2, encoding the synthase and glutaminase subunit of PLP synthase, respectively. In the presence of ammonia, the synthase alone displays an exceptional polymorphic synthetic ability in carrying out a complex set of reactions, including pentose and triose isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization, that convert C3 and C5 precursors into the cofactor B6 vitamer, pyridoxal 5'-phosphate. Here, employing the Bacillus subtilis proteins, we demonstrate key features along the catalytic path. We show that ribose 5-phosphate is the preferred C5 substrate and provide unequivocal evidence that the pent(ul)ose phosphate imine occurs at lysine 81 rather than lysine 149 as previously postulated. While this study was under review, corroborative crystallographic evidence has been provided for imine formation with the corresponding lysine group in the enzyme from Thermotoga maritima (Zein, F., Zhang, Y., Kang, Y.-N., Burns, K., Begley, T. P., and Ealick, S. E. (2006) Biochemistry 45, 14609-14620). We have detected an unanticipated covalent reaction intermediate that occurs subsequent to imine formation and is dependent on the presence of Pdx2 and glutamine. This step most likely primes the enzyme for acceptance of the triose sugar, ultimately leading to formation of the pyridine ring. Two alternative structures are proposed for the chromophoric intermediate, both of which require substantial modifications of the proposed mechanism.     

Biosynthesis of bacterial glycogen. Incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose-protected pyridoxal phosphate binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of allosteric effectors fructose-P2, 5'-AMP, or hexanediol-1,6-P2, protects against pyridoxylation of the allosteric activator site, and allows modification of the ADP-glucose-protected site. Incorporation of pyridoxal-P into the allosteric activator site results in modified enzyme of high activity form, even in the absence of fructose-P2. This modified enzyme, when assayed in the absence of fructose-P2, exhibits activation kinetics similar to nonpyridoxylated enzyme assayed in the presence of fructose-P2 and is still inhibited by 5'-AMP. These data suggest that the allosteric activator site of pyridoxylation is the fructose-P2 binding site, and is distinct from the inhibitor 5'-AMP binding site. Incorporation of pyridoxal-P into the ADP-glucose-protected site results in a decrease in enzyme activity. This pyridoxylated lysine could be involved with the binding of thesubstrates ADP-glucose, alpha-glucose-1-P, or PPi, or participate in the catalytic mechanism of the enzyme.     

Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation

Trehalose is a major storage carbohydrate in budding yeast, Saccharomyces cerevisiae. Alterations in trehalose synthesis affect carbon source-dependent growth, accumulation of glycogen and sporulation. Trehalose is synthesized by trehalose phosphate synthase (TPS), which is a complex of at least four proteins. In this work, we show that the Tps1p subunit protein catalyses trehalose phosphate synthesis in the absence of other TPS components. The tps1-H223Y allele (glc6-1) that causes a semidominant decrease in glycogen accumulation exhibits greater enzyme activity than wild-type TPS1 because, unlike the wild-type enzyme, TPS activity in tps1-H223Y cells is not inhibited by phosphate. Poor sporulation in tps1 null diploids is caused by reduced expression of meiotic inducers encoded by IME1, IME2 and MCK1. Furthermore, high-copy MCK1 or heterozygous hxk2 mutations can suppress the tps1 sporulation trait. These results suggest that the trehalose-6-phosphate inhibition of hexokinase activity is required for full induction of MCK1 in sporulating yeast cells.     

Characterization of an early intermediate in the folding of the alpha subunit of tryptophan synthase by hydrogen exchange measurement

The development of the hydrogen bonding network in the early stages of the folding of the alpha subunit of tryptophan synthase was monitored with a hydrogen exchange technique. The orders of magnitude difference between the rapid conversions of the unfolded forms to two stable intermediates (milliseconds) and the subsequent slow conversions of the intermediates to the native form (greater than 100 s) was used to selectively label with tritium the hydrogen bonds that form in the first 30 s of folding at 0 degree C. Rapid removal of the tritiated solvent by gel filtration ensured that hydrogen bonds formed in subsequent folding reactions would be unlabeled. Limited proteolysis and separation of peptides by high-pressure liquid chromatography permitted the determination of the amount of label retained in individual peptides by scintillation counting. Peptides 1-70 and 71-188, which when covalently linked comprise the stable amino domain in the native conformation, retain 91% and 93%, respectively, of the label retained when the protein is allowed to completely refold in tritiated solvent. Peptide 189-268, the marginally stable carboxyl domain, only retains 43% of the label. The striking difference in retention of label confirms the independent folding of these two domains and shows that the kinetic intermediates that appear in the folding of alpha subunit correspond to structural domains in the native conformation. The near-equality of the labeling of the two peptides comprising the amino domain shows that this domain folds as a single entity and that subdomain folding is unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase

Transfer of potato tubers to low temperature leads after 2–4 d to a stimulation of sucrose synthesis, a decline of hexose-phosphates and a change in the kinetic properties, and the appearance of a new form of sucrose phosphate synthase (SPS). Antisense and co-suppression transformants with a 70–80% reduction in SPS expression have been used to analyse the contribution of SPS to the control of cold sweetening. The rate of sucrose synthesis in cold-stored tubers was investigated by measuring the accumulation of sugars, by injecting labelled glucose of high specific activity into intact tubers, and by providing 50 mol m^–3 labelled glucose to fresh tuber slices from cold-stored tubers. A 70–80% decrease of SPS expression resulted in a reproducible but non-proportional (10–40%) decrease of soluble sugars in cold-stored tubers, and a non-proportional (about 25%) inhibition of label incorporation into sucrose, increased labelling of respiratory intermediates and carbon dioxide, and increased labelling of glucans. The maximum activity of SPS is 50-fold higher than the net rate of sugar accumulation in wild-type tubers, and decreased expression of SPS in the transformants was partly compensated for increased levels of hexose-phosphates. It is concluded that SPS expression per se does not control sugar synthesis. Rather, a comparison of the in vitro properties of SPS with the estimated in vivo concentrations of effectors shows that SPS is strongly substrate limited in vivo . Alterations in the kinetic properties of SPS, such as occur in response to low temperature, will provide a more effective way to stimulate sucrose synthesis than changes of SPS expression.     

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.     

Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from Plasmodium falciparum

Pyridoxal 5′-phosphate (PLP) is required as a cofactor by many enzymes. The predominant de novo biosynthetic route is catalyzed by a heteromeric glutamine amidotransferase consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. Previously, Bacillus subtilis PLP synthase was studied by X-ray crystallography and complex assembly had been characterized by isothermal titration calorimetry. The fully assembled PLP synthase complex contains 12 individual Pdx1/Pdx2 glutamine amidotransferase heterodimers. These studies revealed the occurrence of an encounter complex that is tightened in the Michaelis complex when the substrate l-glutamine binds. In this study, we have characterized complex formation of PLP synthase from the malaria-causing human pathogen Plasmodium falciparum using isothermal titration calorimetry. The presence of l-glutamine increases the tightness of the interaction about 30-fold and alters the thermodynamic signature of complex formation. The thermodynamic data are integrated in a 3D homology model of P. falciparum PLP synthase. The negative experimental heat capacity (C_p) describes a protein interface that is dominated by hydrophobic interactions. In the absence of l-glutamine, the experimental C_p is less negative than in its presence, contrasting to the previously characterised bacterial PLP synthase. Thus, while the encounter complexes differ, the Michaelis complexes of plasmodial and bacterial systems have similar characteristics concerning the relative contribution of apolar/polar surface area. In addition, we have verified the role of the N-terminal region of PfPdx1 for complex formation. A “swap mutant” in which the complete αN-helix of plasmodial Pdx1 was exchanged with the corresponding segment from B. subtilis shows cross-binding to B. subtilis Pdx2. The swap mutant also partially elicits glutaminase activity in BsPdx2, demonstrating that formation of the protein complex interface via αN and catalytic activation of the glutaminase are linked processes.     

An obligatory intermediate controls the folding of the alpha-subunit of tryptophan synthase, a TIM barrel protein

The proposed kinetic folding mechanism of the alpha-subunit of tryptophan synthase (alphaTS), a TIM barrel protein, displays multiple unfolded and intermediate forms which fold through four parallel pathways to reach the native state. To obtain insight into the secondary structure that stabilizes a set of late, highly populated kinetic intermediates, the refolding of urea-denatured alphaTS from Escherichia coli was monitored by pulse-quench hydrogen exchange mass spectrometry. Following dilution from 8 M urea, the protein was pulse-labeled with deuterium, quenched with acid and mass analyzed by electrospray ionization mass spectrometry (ESI-MS). Hydrogen bonds that form prior to the pulse of deuterium offer protection against exchange and, therefore, retain protons at the relevant amide bonds. Consistent with the proposed refolding model, an intermediate builds up rapidly and decays slowly over the first 100 seconds of folding. ESI-MS analysis of the peptic fragments derived from alphaTS mass-labeled and quenched after two seconds of refolding indicates that the pattern of protection of the backbone amide hydrogens in this transient intermediate is very similar to that observed previously for the equilibrium intermediate of alphaTS highly populated at 3 M urea. The protection observed in a contiguous set of beta-strands and alpha-helices in the N terminus implies a significant role for this sub-domain in directing the folding of this TIM barrel protein.