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)

Zinc and calcium ions play important roles in the biosynthesis and storage of insulin. Insulin biosynthesis occurs within the beta-cells of the pancreas via preproinsulin and proinsulin precursors. In the golgi apparatus, proinsulin is sequestered within Zn(2+)- and Ca(2+)-rich storage/secretory vesicles and assembled into a Zn(2+) and Ca(2+) containing hexameric species, (Zn(2+))(2)(Ca(2+))(Proin)(6). In the vesicle, (Zn(2+))(2)(Ca(2+))(Proin)(6) is converted to the insulin hexamer, (Zn(2+))(2)(Ca(2+))(In)(6), by excision of the C-peptide through the action of proteolytic enzymes. The conversion of (Zn(2+))(2)(Ca(2+))(Proin)(6)to (Zn(2+))(2)(Ca(2+))(In)(6) significantly lowers the solubility of the hexamer, causing crystallization within the vesicle. The (Zn(2+))(2)(Ca(2+))(In)(6) hexamer is an allosteric protein that undergoes ligand-mediated interconversion among three global conformation states designated T(6), T(3)R(3) and R(6). Two classes of allosteric sites have been identified; hydrophobic pockets (3 in T(3)R(3) and 6 in R(6)) that bind phenolic ligands, and anion sites (1 in T(3)R(3) and 2 in R(6)) that bind monovalent anions. The allosteric states differ widely with respect to the physical and chemical stability of the insulin subunits. Fusion of the vesicle with the plasma membrane results in the expulsion of the insulin crystals into the intercellular fluid. Dissolution of the crystals, dissociation of the hexamers to monomer and transport of monomers to the liver and other tissues then occurs via the blood stream. Insulin action then follows binding to the insulin receptors. The role of Zn(2+) in the assembly, structure, allosteric properties, and dynamic behavior of the insulin hexamer will be discussed in relation to biological function.

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the UV-visible absorption changes (300-550 nm) that occur in the spectrum of enzyme-bound pyridoxal 5'-phosphate during the reaction of L-serine with the alpha 2 beta 2 and beta 2 forms of Escherichia coli tryptophan synthase. In agreement with previous kinetic studies [Lane, A., & Kirschner, K. (1983) Eur. J. Biochem. 129, 561-570], the reaction with alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3). The RSSF data reveal that during tau 1, the internal aldimine, E(PLP), with lambda max = 412 nm (pH 7.8), undergoes rapid conversion to two transient species, one with lambda max congruent to 420 nm and one with lambda max congruent to 460 nm. These species decay in a biphasic process (1/tau 2, 1/tau 3) to a complicated final spectrum with lambda max congruent to 350 nm and with a broad envelope of absorbance extending out to approximately 525 nm. Analysis of the time-resolved spectra establishes that the spectral changes in tau 2 are nearly identical with the spectral changes in tau 3. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to increase the amount of the 420-nm transient and to decrease the amount of the species with lambda max congruent to 460 nm. These findings identify the serine Schiff base (the external aldimine) as the 420 nm absorbing, highly fluorescent transient; the species with lambda max congruent to 460 nm is the delocalized carbanion (quinoidal) species derived from abstraction of the alpha proton from the external aldimine. The reaction of L-serine with beta 2 consists of two relaxations (1/tau 1 beta greater than 1/tau 2 beta) and yields a quasi-stable species with lambda max = 420 nm, in good agreement with a previous report [Miles, E. W., Hatanaka, M., & Crawford, I. P. (1968) Biochemistry 7, 2742-2753]. Analysis of the RSSF spectra indicates that the same spectral change occurs in each phase of the reaction. The similarity of the spectral changes that occur in tau 2 and tau 3 of the alpha 2 beta 2 reaction is postulated to originate from the existence of two (slowly) interconverting forms of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

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The chromophoric divalent metal ion chelators 4-(2-pyridylazo)resorcinol (PAR) and 2,2',2"-terpyridine (terpy) are used as kinetic and spectroscopic probes to investigate in solution the SCN- -induced conformational transformations of the insulin, proinsulin, and miniproinsulin hexamers (miniproinsulin is a proinsulin analogue wherein the C-chain is replaced by a dipeptide cross-link between Gly-A1 and Ala-B30). Herein we designate the 2Zn and 4Zn crystal forms of the hexamer as the T6 and T3R3 conformations, respectively. For all three proteins, addition of SCN- reduces the rate of sequestering and removal of zinc ion by chelator. The effect of SCN- on the rate of this process saturates at the same concentration (30 mM) known to induce the T6 to T3R3 transformation in the insulin crystal. Under both T6 and T3R3 conditions, the critical stoichiometry for high-affinity interaction between Zn2+ and each of the three proteins is shown to be 2 mol of Zn2+/mol of protein hexamer. Consequently, we confirm the finding that off-axial coordination of Zn2+ via His-B10 and His-B5 residues is of minor importance for the SCN- -induced conformation change in solution [Renscheidt, H., Strassburger, W., Glatter, U., Wollmer, A., Dodson, G. G., & Mercola, D. A. (1984) Eur. J. Biochem. 142, 7-14]. Under T6 conditions, the kinetics of the reactions between insulin, proinsulin, and miniproinsulin and a variable excess of terpy are similar and biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)

The tryptophan synthase α2β2 bi-enzyme complex catalyzes the last two steps in the synthesis of L-tryptophan (L-Trp). The α-subunit catalyzes cleavage of 3-indole-D-glycerol 3’-phosphate (IGP) to give indole and D-glyceraldehyde 3’-phosphate (G3P). Indole is then transferred (channeled) via an interconnecting 25 Å-long tunnel, from the α-subunit to the (β-subunit where it reacts with L-Ser in a pyridoxal 5’-phosphate-dependent reaction to give L-Trp and a water molecule. The efficient utilization of IGP and L-Ser by tryptophan synthase to synthesize L-Trp utilizes a system of allosteric interactions that (1) function to switch the α-site on and off at different stages of the β-subunit catalytic cycle, and (2) prevent the escape of the channeled intermediate, indole, from the confines of the α- and β-catalytic sites and the interconnecting tunnel. This review discusses in detail the chemical origins of the allosteric interactions responsible both for switching the α-site on and off, and for triggering the conformational changes between open and closed states which prevent the escape of indole from the 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)

The glyoxylate cycle is an anaplerotic pathway of the tricarboxylic acid (TCA) cycle that allows growth on C 2 compounds by bypassing the CO 2 -generating steps of the TCA cycle. The unique enzymes of this route are isocitrate lyase (ICL) and malate synthase (MS). ICL cleaves isocitrate to glyoxylate and succinate, and MS converts glyoxylate and acetyl-CoA to malate. The end products of the bypass can be used for gluconeogenesis and other biosynthetic processes. The glyoxylate cycle occurs in Eukarya, Bacteria and Archaea. Recent studies of ICL-and MS-deficient strains as well as proteomic and transcriptional analyses show that these enzymes are often important in human, animal and plant pathogenesis. These studies have extended our understanding of the metabolic pathways essential for the survival of pathogens inside the host and provide a more complete picture of the physiology of pathogenic micro-organisms. Hopefully, the recent knowledge generated about the role of the glyoxylate cycle in virulence can be used for the development of new vaccines, or specific inhibitors to combat bacterial and fungal diseases.