John K. Hardman

Substrate interactions with the α-subunit of the Escherichia coli tryptophan synthase: A kinetic study of the wild-type α-subunit☆

Abstract The kinetics of the reversible reaction [indoleglycerol phosphate (InGP) ⇌ indole and glyceraldehyde 3-phosphate] catalyzed by the α-subunit of the Escherichia coli tryptophan synthase was examined. The forward reaction (InGP to indole) is characterized by simple Michaelis-Menton kinetics at low ( m ) InGP concentrations and is consistent with the binding of a single InGP molecule at the active site. At higher (>2 m m ) InGP concentrations, there is a very sharp decrease in rate. This concentration-dependent rate decrease parallels a sharp concentration-dependent aggregation of InGP. This rate decrease at high InGP concentrations may be due partially to this aggregation and the consequent lowering of monomeric InGP concentration. The reverse reaction (indole to InGP) is characterized by a sigmoidal response of rate of indole concentration. The data (Hill plot and double reciprocal 1 v versus 1/[indole] 2 plot) are consistent with two strongly interacting sites, an effector site and the active site. The effect of InGP on the reverse reaction is InGP concentration dependent. At low InGP concentrations, there is an inhibition with a concomitant slight decrease in the V . Under these conditions, the two indole sites are still apparent. These data are interpreted as being due to monomeric InGP binding only to the glyceraldehyde 3-phosphate portion of the active site. At high InGP concentrations, there is an increased activation of the reverse reaction and nearly a doubling in V . In addition, the Hill slope approaches a value of 2 and the double-reciprocal plot ( 1 v versus 1/[indole] 2 ) is linear only at high indole concentrations. These data are consistent with the interpretation that InGP (probably the aggregate form) binds to a separate effector site and that this binding precludes the functioning of the indole effector site at low indole concentrations. The effect of 1 or 7 m m indole on the forward reaction is negligible or slightly stimulatory. At low InGP concentrations, the effect of indole may be due to a diminished activation of the active site when it is occupied by InGP rather than when it is occupied by indole and glyceraldehyde 3-phosphate. At high InGP concentrations, the effect of indole can be attributed to the displacement of InGP by indole in the InGP aggregate.

Chemical and ultrastructural studies on the cell wall ofStaphylococcus simulans biovarstaphylolyticus

Cell walls of strains ofStaphylococcus simulans biovarstaphylolyticus andS. aureus FDA 209P were compared ultrastructurally and chemically to investigate the mechanism of resistance of this strain ofS. simulans to its own staphylolytic endopeptidase. Chemical analysis of the peptidoglycans of the various strains examined showed that cells that were more resistant to lysis by the endopeptidase had lower glycine/serine ratios in their cross bridges and that, within a species, the more resistant cells had either fewer residues in these cross bridges or fewer cross bridges. Ultrastructural studies showed that cell wall thickness was not involved in resistance to the enzyme. Comparisons of the endopeptidase susceptibility of intact cells and isolated peptidoglycans from these cells suggested that the three-dimensional structure of the cell wall may play a role in resistance to lysis by the endopeptidase.

FLAVIN AS AN ACTIVE COMPONENT OF THE PHOTOREVERSIBLE PIGMENT SYSTEM OF THE GREEN ALGA PROTOSIPHON BOTRYOIDES KLEBS

Abstract— The photoreversible pigment system from Protosiphon botryoides Klebs can be separated into two fractions, which are individually inactive photobiologically, but can be recombined to form an active system. One fraction contains a component stable to combined heat and dilute acid treatment; this component can be replaced by flavin nucleotides. An essential component in the second fraction is heat labile. A similar pigment system has been isolated from other species of green algae.

FLAVIN COMPOUNDS AS AGENTS FOR THE OXIDATION OF PLASTOCYANIN IN BLUE LIGHT

Abstract—Flavins, flavin nucleotides and selected flavoproteins have been compared in a reaction using blue light. in which plastocyanin is oxidized as the flavin is photoreduced. Per unit of light absorbed. flavin mononucleotide is more effective than flavin adenine dinucleotide, ribotlavin or lumiflavin. Of the flavoproteins tested, diaphorase from Clostridium kluyveri was most effective, but was less active than free flavin mononucleotide. The oxidation of plastocyanin requires aerobic conditions. and appears to be mediated by the production of singlet oxygen when the flavin is irradiated.

A BLUE LIGHT REACTION INVOLVING FLAVTN NUCLEOTIDES AND PLASTOCYANIN FROM PROTOSIPHON BOTRYOIDES

Abstract— A pigment that absorbs yellow light (580 nm) appears in a cell‐free preparation of Protosiphon when it is irradiated with blue light (430 nm) in the presence of flavoprotein from Photosiphon or flavin nucleotides. The pigment has been isolated and purified. It is a protein with a molecular weight of approximately 9000 daltons. An amino acid analysis reveals that it lacks arginine and tryptophan. It is colorless when reduced and blue (absorbs at 580 nm) when oxidized. Its normal oxidation–reduction potential is + 0.37 V. It contains one copper ion per molecule and has other properties that match those of plastocyanin isolated from green cells of other species.

Tryptophan-containing -Subunits of the Escherichia coli Tryptophan Synthase ENZYMATIC AND UREA STABILITY PROPERTIES

Early studies suggested that the Escherichia coli tryptophan synthase α-subunit unfolded in a two-step process in which there was a stable intermediate composed of a native α-1 folding unit (residues 1-188) and a completely unfolded α-2 folding unit (residues 189-268). More recent evidence has indicated that such a structure for the intermediate seems unlikely. In this report, single Trp residues (absent in the wild-type α-subunit) are substituted separately for Phe residues at positions 139 (in α-1) and 258 (in α-2) to produce the F139W, F258W, and F139W/F258W mutant α-subunits. The UV absorbance and fluorescence properties of the F139W/F258W double mutant are identical with those of equimolar mixtures of the single mutants, suggesting that the Trp residue at each position can independently report the behavior of its respective folding unit. Each mutant α-subunit is wild-type enzymatically, and when UV absorbance is monitored, the urea-induced unfolding of the three tryptophan-containing α-subunits is virtually identical to the wild-type protein. These wild-type properties make these proteins attractive candidates for a fluorescence examination of the behavior of the individual folding units and the structure of potential intermediate(s) and as host proteins for the insertion of our existing destabilizing and/or stabilizing mutational alterations.

Substrate interactions with the α-subunit of the Escherichia coli tryptophan synthase: A study of the activity of mutant α-subunits☆

Abstract Extracts of 19 trpA mutant strains of Escherichia coli were examined for their relative activity in the reversible aldolytic reaction catalyzed by the trpA gene product, the α-subunit of tryptophan synthase, in combination with the β-subunit of this enzyme. The specific activities in this reaction, indoleglycerol-P (InGP) ⇌ indole + glyceraldehyde-3-P, were determined for both the forward reaction (InGP to indole) and the reverse reaction (indole to InGP). The majority of the mutant α-subunits had 50% of the wild-type activity in the reverse reaction. Several had 5 to 15% of wild-type specific activity in the forward reaction but 60 to 100% of wild-type specific activity in the reverse reaction. Spontaneous revertant strains, selected for their increased ability to catalyze the forward reaction effectively, contained α-subunits with the expected higher specific activities in the forward reaction but without parallel changes in the reverse reaction activity.

Photoaffinity labeling of the indole sites on the Escherichia coli tryptophan synthase α-subunit☆

Abstract The α subunit of the Escherichia coli tryptophan synthase catalyzes the reversible aldolytic reaction: Indole-3-glycerol phosphate ⇌ indole + glyceraldehyde 3-phosphate. The use of 5-azidoindole as a photoaffinity label has made the generation of a number of enzyme-substrate complexes possible, each with a given degree of saturation of the two postulated indole sites. When assayed in the reverse reaction (indole-3-glycerol phosphate synthesis), samples of α subunit treated at concentrations of 5-azidoindole less than or equal to 2 m m show a progressive 30–40% activation. A gradual inactivation occurs only in samples irradiated at concentrations in excess of 2 m m 5-azidoindole, and this inactivation is complete at 8–10 m m . A quantitatively similar activation occurs in the forward reaction (indole synthesis), however inactivation in this case is incomplete, with complexes treated at 8–12 m m 5-azidoindole retaining 30–40% relative activity in this reaction. When treated α subunits were assayed for their abilities to complement the β2-subunit in the reactions indole + l -serine → l -tryptophan + H2O and indole-3-glycerol phosphate + l -serine → l -tryptophan + glyceraldehyde 3-phosphate, quantitatively lesser amounts of activation followed by total inactivation are observed over a similar range of 5-azidoindole concentrations.

5-Azidoindole binding to the Escherichia coli tryptophan synthase α2β2 complex☆

Abstract The tryptophan synthase α 2 β 2 complex catalyzes tryptophan (Trp) biosynthesis from serine plus either indole (IN) or indole-3-glycerol phosphate (InGP). The photoreactive 5-azido analog in IN (AzIN), itself a substrate in the dark, was utilized to examine the substrate binding sites on this enzyme. When irradiated with AzIN at concentrations approaching IN saturation for the IN → Trp activity (0.1 m m ), in the absence of serine, the enzyme was increasingly inactivated (up to 70–80%) concomitant with the progressive binding of a net of 2 mol AzIN per αβ equivalent. Little or no cooperativity in the binding of the 2 mol AzIN was observed. In contrast, there was minimal effect on the IN → InGP activity. Under these conditions AzIN appeared to be incorporated equally into each subunit. No significant inactivation nor binding occurred in the presence of serine. A quantitatively similar inactivation of InGP → Trp activity was observed over the same AzIN concentration range, suggesting common IN sites for Trp biosynthesis from either indole substrate. At higher concentrations (0.1–0.7 m m ), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/αβ equivalent). These data are consistent, although more clear-cut quantitatively, with the high- and low-affinity sites proposed from equilibrium dialysis studies. AzIN binding studies utilizing the isolated β 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit.