13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein

13C NMR has been used to observe the equilibrium complex of [4-13C]-5-aminolevulinate ([4-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [4-13C]ALA (chemical shift = 205.9 ppm) forms [3,5-13C]PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of [4-13C]ALA and [15N]ALA was used to assign the 121.0 and 123.0 ppm resonances to C5 and C3, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and [4-13C]ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approximately 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of less than 10 s-1, which is consistent with the turnover rate of the enzyme. For the complex formed from [4-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift = 166.5 ppm, line width approximately 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with 113Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn(II) enzyme. Neither splitting nor broadening from 113Cd-13C coupling was observed.     

5-Chlorolevulinate modification of porphobilinogen synthase identifies a potential role for the catalytic zinc.

Porphobilinogen synthase (PBGS) is a Zn(II) metalloenzyme which catalyzes the asymmetric condensation of two molecules of 5-aminolevulinate (ALA). The nitrogen of the first substrate ends up in the pyrrole ring of product (P-side ALA); by contrast, the nitrogen of the second substrate molecule remains an amino group (A-side ALA). A reactive mimic of the substrate molecules, 5-chlorolevulinate (5-CLA), has been prepared and used as an active site directed irreversible inhibitor of PBGS. Native octameric PBGS binds eight substrate molecules and eight Zn(II) ions, with two types of sites for each ligand. As originally demonstrated by Seehra and Jordan [(1981) Eur. J. Biochem. 113, 435-446], 5-CLA inactivates the enzyme at the site where one of the two substrate molecules binds, and modification at four sites per octamer (one per active site) affords near-total inactivation. Here we report that 5-CLA-modified PBGS (5-CLA-PBGS) can bind up to four substrate molecules and four Zn(II) ions. Contrary to the conclusion of Seehra and Jordan, we find that the preferential site of 5-CLA inactivation is the A-side ALA binding site. On the basis of the dissociation constants, the metal ion binding sites lost upon 5-CLA modification are assigned to the four catalytic Zn(II) sites. 5-CLA-PBGS is shown to be modified at cysteine-223 on half of the subunits. We conclude that cysteine-223 is near the amino group of A-side ALA and propose that this cysteine is a ligand to the catalytic Zn(II). The vacant substrate binding site on 5-CLA-PBGS is that of P-side ALA. We have used 13C and 15N NMR to view [4-13C]ALA and [15N]ALA bound to 5-CLA-PBGS. The NMR results are nearly identical to those obtained previously for the enzyme-bound P-side Schiff base intermediate [Jaffe et al. (1990) Biochemistry 29, 8345-8350]. It appears that, in the absence of the catalytic Zn(II), 5-CLA-PBGS does not catalyze the condensation of the amino group of the P-side Schiff base intermediate with the C4 carbonyl derived from 5-CLA. On this basis we propose that Zn(II) plays an essential role in formation of the first bond between the two substrate molecules.     

13C NMR studies of methylene and methine carbons of substrate bound to a 280,000-dalton protein, porphobilinogen synthase

13C NMR has been used to observe the equilibrium complex of [5,5-2H,5-13C]-5-aminolevulinate [( 5,5-2H,5-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [5,5-2H,5-13C]ALA (chemical shift 46.9 ppm in D2O) was prepared from [5-13C]ALA through enolization in deuteriated neutral potassium phosphate buffer. In the PBG synthase reaction [5,5-2H,5-13C]ALA forms [2,11,11-2H,2,11-13C]PBG (chemical shifts 116.2 ppm for C2 and 34.2 ppm for C11 in D2O). For the complex formed between [5,5-2H,5-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation but can form a Schiff base adduct, the chemical shift of 44.2 ppm (line width 92 Hz) identifies an imine structure as the predominant tautomeric form of the Schiff base. By comparison to model compounds, the stereochemistry of the imine has been deduced; however, the protonation state of the imine nitrogen remains unresolved. Reconstitution of the MMTS-modified enzyme-Schiff base complex with Zn(II) and 2-mercaptoethanol results in the holoenzyme-bound equilibrium complex; this complex contains predominantly enzyme-bound PBG, and spectra reveal two peaks from bound PBG and two from free PBG. For bound PBG, C2 is -2.8 ppm from the free signal and C11 is +2.6 ppm from the free signal; the line widths of the bound signals are 55 and 75 Hz, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the biosynthesis of cyclitols, XXXV. On the mechanism of action of myo-inositol-1-phosphate synthase from rat testicles.

The animal myo-inositol-1-phosphate synthase is competitively inhibited by pyridoxal phosphate and trinitrobenzensulphonate, both compounds known to prevent Schiff's base formation. When incubated with labelled substrate and then treated with NaBH4, label can be recovered in the enzyme protein. In analogous experiments with tritiated NaBH4 the enzyme protein also becomes labelled; after hydrolysis of such protein only one labelled compound, derived from lysine and D-glucose 6-phosphate, could be isolated. Its exact structure is not yet known. From these results it can be concluded that during its action myo-inositol-1-phosphate synthase forms a Schiff's base with its substrate, in analogy to the class I aldolases.     

Dehydroquinate synthase: the role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis

The cofactor requirements of dehydroquinate synthase from Escherichia coli have been characterized. The homogeneous enzyme, purified from the overproducing strain RB791 (pJB14), is a monomeric metalloenzyme of Mr = 39,000 that contains 1 mol of tightly bound Co(II) according to atomic absorption analysis. The holoenzyme rapidly loses activity upon incubation with EDTA, giving rise to a stable but catalytically inactive apoenzyme. Activity is fully restored by reconstitution with Co(II) and partially restored with other divalent cations. Reconstitution of the apoenzyme with Zn(II) (which is probably the functioning metal in vivo) restores activity to 53% of the level observed with the Co(II)-holoenzyme. The presence of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) blocks the inactivation by EDTA. Dehydroquinate synthase also binds 1 mol of NAD+, the presence of which is essential for catalytic activity. The rate constant for the dissociation of NAD+ from the Co(II)-holoenzyme was found to be 0.024 min-1. Under turnover conditions with saturating levels of substrate, the dissociation rate of NAD+ increases by a factor of 40, to 1 min-1. Under these conditions (pH 7.5, 20 degrees C), the Km for NAD+ was determined to be 80 nM.     

15N and 13C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate

Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through 13C and 15N NMR. To our knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by 13C or 15N NMR. Here we extend our 13C NMR studies to PBGS complexes with [3,3-2H2,3-13C]ALA and report 15N NMR studies of [15N]ALA bound to PBGS. As in our previous 13C NMR studies, observation of enzyme-bound 15N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pKa is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent (kexchange greater than 10(2) s-1). For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C4 of ALA and an active-site lysine. The 13C chemical shift of [3,3-2H2,3-13C]ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between [15N]ALA and hydrazine or hydroxylamine, the 15N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation; again the protons are in rapid exchange with solvent. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C4 of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.     

L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate synthase

L-Vinylglycine (L-VG) has been shown to be a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate (ACC) synthase [Satoh, S., and Yang, S. F. (1989) Plant Physiol. 91, 1036-1039] as well as of other pyridoxal phosphate-dependent enzymes. This report demonstrates that L-VG is primarily an alternative substrate for the enzyme. The L-VG deaminase activity of ACC synthase yields the products alpha-ketobutyrate and ammonia with a k(cat) value of 1.8 s(-1) and a K(m) value of 1.4 mM. The k(cat)/K(m) of 1300 M(-1) s(-1) is 0.17% that of the diffusion-controlled reaction with the preferred substrate, S-adenosyl-L-methionine. The enzyme-L-VG complex partitions to products 500 times for every inactivation event. The catalytic mechanism proceeds through a spectrophotometrically detected quinonoid with lambda(max) of 530 nm, which must rearrange to a 2-aminocrotonate aldimine to yield final products. Alternative mechanisms for the inactivation reaction are presented, and the observed kinetics for the full reaction course are satisfactorily modeled by kinetic simulation. The inactive enzyme is an aldimine with lambda(max) of 432 nm. It is resistant to NaBH(3)CN but is reduced by NaBH(4). ACC synthase is now expressed in Pichia pastoris with an improved yield of 10 mg/L.     

Studies on the biosynthesis of cyclitols, XXXVII. On mechanism and function of Schiff's base formation as an intermediary reaction step of myo-inositol-1-phosphate synthase from rat testicles.

It could be shown that the formation of a Schiff's base by myo-inositol-1-phosphate synthase of rat testicles occurs by binding the aldehyde group of the open form of its substrate, D-glucose 6-phosphate, to a lysyl residue of one or both smaller subunits of the enzyme. The participation of the Schiff's base formation in the catalytic process is supported by the observations that (a) no Schiff's base is formed if NAD is removed from the enzyme, and (b) in the presence of NAD, the dehydrogenation step involved in the catalytic mechanism apparently takes place rapidly after the formation of the Schiff's base.     

Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase

Rat methionine synthase was expressed chiefly as apoenzyme in recombinant baculovirus-infected insect cells (Yamada, K., Tobimatsu, T., and Toraya, T. (1998) Biosci. Biotech. Biochem. 62, 2155-2160). The apoenzyme produced was very unstable, and therefore, after complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K(m) values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob(II)alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium(III) and S-adenosylmethionine. The rate of oxidative inactivation of the enzyme in the absence of S-adenosylmethionine was slower with a stronger reducing agent like titanium(III). The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective or were effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme, which was convertible to holoenzyme by photoirradiation.     

Increased 5-aminolevulinic acid (ALA) in heavy metal exposed bivalve molluscs,Cerastoderma edule: An effect due to inhibition of porphobilinogen synthase?

1. The effect of cadmium, lead, and mercury on 5-aminolevulinic acid (ALA), porphobilinogen (PBG), and PBG synthase was determined in hepatopancreas of the bivalve, Cerastoderma edule (L.).2. Cd and Hg exposure induced increased ALA content, and thus an initial doubling of ALA within 24 hr.3. Using ALA in excess (8 mmoll⁻¹) as substrate, no PBG synthase (ALA dehydratase, EC 4.2.1.24) activity was detectable in freshly prepared hepatopancreas homogenates.4. Increased ALA in metal exposed bivalves is not a simple effect due to metal inhibition of PBG synthase.5. The observed lack of PBG synthase suggests an alternative to the general pathway where two ALA molecules condense to one PBG.     

Enzyme elements involved in the interconversion of L-carbamylaspartate and L-dihydroorotate by dihydroorotase from Clostridium oroticum

Enzyme elements that are involved in the reversible cyclization of L-carbamylaspartate to L-dihdroorotate catalyzed by dihydroorotase (EC 3.5.2.3) from Clostridium oroticum (ATCC 25750) have been studied. Removal of Zn(II) from the enzyme by chelators followed by incubation of apoenzyme with Co(II) results in replacement of two to three of the four Zn(II) ions per molecule by Co(II). The catalytic properties of the Zn(II)Co(II) dihydroorotase are different from those of native enzyme. The Vmax is increased for both the synthesis and hydrolysis of L-dihydroorotate. The Km for L-dihydroorotate is unchanged, while the Km for L-carbamylaspartate is increased more than twofold. On the other hand, the kinetic properties of Zn(II)-reconstituted dihydroorotase are indistinguishable from those of native enzyme. The pH dependence of Vmax is also altered by the Co(II) substitution. For both Zn(II)- and Zn(II)Co(II)-dihydroorotase, this pH dependence is well described by a single ionization and the pK's for L-dihydroorotate synthesis and hydrolysis are different. Substitution with Co(II) increases the pK for both reaction directions to different extents. These results strongly support a role for the tightly bound metals in the catalytic mechanism. In addition, diethylpyrocarbonate rapidly inactivates the enzyme. The inactivation is prevented by L-dihydroorotate. This result is consistent with a role for at least one histidine in catalysis. The possibility that C. oroticum dihydroorotase may be useful model for the more complex mammalian enzyme is considered.     

Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes.

A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285 000) is made up of eight apparently identical subunits of Mr 35 000. The enzyme is sensitive to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (approximately equal to 12% of control) when assayed in buffers devoid of Zn2+. Addition of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 microM-Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constant for E X S X Pb2+ complex) = 25.3 +/- 3.0 microM; KiS (dissociation constant for E X Pb2+ complex) = 9.0 +/- 2.0 microM]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.     

Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.

The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.     

Is the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tyrosine sensitive), a copper metalloenzyme?

3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (tyrosine sensitive) was purified from Escherichia coli carrying the plasmid pKB45. Enzyme of high specific catalytic activity (70 mu/mg) was obtained from cells grown only to the early log phase. The purified protein contained Cu(II) and showed an absorption band at 350 nm. Metal-free, catalytically inactive apoenzyme could be produced by dialysis against cyanide ion, and the holoenzyme could be reconstituted in terms of both catalytic activity and A350 by the binding of one Cu(II) ion per enzyme subunit. Zn(II) also reactivated the apoenzyme to about 50% of the level seen with Cu(II), although in this case no band appeared at 350 nm. In contrast to earlier reports that the enzyme contains substoichiometric levels of iron, insignificant amounts of iron were found in the isolated enzyme, and neither Fe(II) nor FE(III) regenerated either an absorption band at 350 nm or any catalytic activity from the apoenzyme. The evident preference of the enzyme as isolated for (Cu)II suggests that the synthase might naturally be a copper metalloenzyme.     

Mechanistic implications of mutations to the active site lysine of porphobilinogen synthase

Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices. (13)C NMR studies of [3,5-(13)C]porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e. environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coli PBGS and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.     

Inactivation of dioldehydrase in the presence of a coenzyme-B12 analog

We have previously shown that a coenzyme-B₁₂ analog, adenosylcobalamin (AdoCbl)-(e-OH), with the e-propionamide group converted to a carboxylic acid, serves as a poor coenzyme for dioldehydrase. During the course of the catalytic process, the enzyme AdoCbl-(e-OH) complex becomes catalytically inactive (T. Toraya, E. Krodel, A. S. Mildvan, and R. H. Abeles, 1979, Biochemistry18, 417–426). We have now examined the mechanism of this inactivation further. Inactivation only occurs in the presence of substrate. The dioldehydrase coenzyme analog complex is stable in the absence of substrate. In the inactivated complex, the coenzyme analog was stoichiometrically converted to a cob(II)alamin species. The cob-(II)alamin formed remained irreversibly bound at the active site of the enzyme and resisted oxidation by O₂ even in the presence of CN^?. Stoichiometric formation of 5′-deoxyadenosine from the 5′-deoxy-5′-adenosyl moiety of the coenzyme analog was demonstrated with [8-¹⁴C]-AdoCbl(e-OH). This nucleoside also remained tightly bound to the enzyme and was not exchangeable with free 5′-deoxyadenosine nor was it removed by Sephadex chromatography. The rate of inactivation showed no deuterium isotope effect when the inactivation occurred in the presence of l,2-propanediol-l-d₂. The inactivated complex was resolved by acid ammonium sulfate treatment into the intact apoenzyme and the hydroxocobalamin derivative. This indicates that the apoenzyme itself is not modified in the inactivation process. These results suggest that the inactivation reaction occurs from one of the intermediates in the normal catalysis. We propose that the inactivation is due to incorrect binding of the modified coenzyme in an intermediate of the catalytic process. This incorrect binding leads to the loss of the substrate radical, and consequently, to loss of catalytic activity.     

Iron extraction from soybean lipoxygenase 3 and reconstitution of catalytic activity from the apoenzyme

Lipoxygenases contain a unique nonheme iron cofactor with a redox role in the catalyzed reaction. The conditions for the extraction of the metal atom were investigated for one of the soybean lipoxygenase isoenzymes. Removal of the iron by o-phenanthroline was attained in the presence of substrate under anaerobic conditions, but the apoenzyme could not be isolated and reconstituted. The freshly regenerated sodium form of Chelex-100 also removes the iron atom from native soybean lipoxygenase 3, but only in sodium bicarbonate buffer at pH 8.0. The soluble but inactive apoenzyme was reconstituted with ferric ammonium sulfate in Tris--HCl buffer at pH 7.0. Stoichiometric iron in the reconstituted enzyme was established using inductively coupled plasma-atomic emission spectroscopy. The reconstituted enzyme contained 90 +/- 10% of the specific activity of the native enzyme. The native configuration of the reconstituted iron site was confirmed by electron paramagnetic resonance spectroscopy.     

Chlorophyll biosynthesis in Chlamydomonas starts with the formation of glutamyl-tRNA

An RNA moiety has been shown to be involved in the conversion of Glu to delta-aminolevulinic acid (ALA), the first committed intermediate of the chlorophyll pathway. We now have evidence suggesting that in Chlamydomonas, the first reaction for converting Glu to ALA is the aminoacylation of Glu to a Glu-specific tRNA. The Glu-tRNA thus formed could be the substrate for Glu-1-semialdehyde synthesis catalyzed by a postulated dehydrogenase. Glu-1-semialdehyde can be converted to ALA by an aminotransferase. Of the three reactions converting Glu to ALA, only the second reaction, catalyzed by a postulated dehydrogenase, is sensitive to inhibition by heme (a known inhibitor of ALA synthesis). We think the regulated enzyme of ALA synthesis is the postulated dehydrogenase. It is postulated that in the chloroplast of Chlamydomonas, the synthesis of ALA and the synthesis of proteins may share a common pool of glutamyl-tRNA.     

Affinity labeling of spinach leaf phosphoribulokinase by ATP analogs. Modification of an active site lysine

Spinach leaf phosphoribulokinase is sensitive to modification by ATP analogs that react with lysine residues. The 2',3'-dialdehyde derivative of ATP (oATP) inactivates enzyme in a slow, time-dependent fashion. The process follows first-order kinetics (kinact = 0.07 min-1), and the concentration dependence of inactivation indicates tight inhibitor binding (Ki = 106 microM). ATP offers good protection against inactivation (Kd = 67 microM), suggesting that oATP is directed toward the catalytic site. This conclusion is supported by the fact that oATP functions as an alternate substrate (Km = 0.55 mM). Inactivation of phosphoribulokinase by [14C]oATP results in a modification stoichiometry of 0.7/site. The 14C-labeled enzyme is stable to dialysis, suggesting that the covalent adduct formed between protein and oATP is not a simple Schiff's base. Adenosine di- and triphosphopyridoxals (Ado-P2-Pl and Ado-P3-Pl, respectively) also inhibit spinach phosphoribulokinase in a time-dependent fashion. In this case, activity loss is reversible unless the inhibited species is borohydride-reduced, suggesting that Ado-P2-Pl and Ado-P3-Pl form Schiff's bases with an amino group on the enzyme. Protection is afforded by the substrate ATP, suggesting that modification is active site-directed. Prolonged incubation of enzyme with these inhibitors does not result in complete inactivation of phosphoribulokinase. Residual activity is dependent on inhibitor concentration, as would be expected if equilibrium is established between the noncovalent E.I complex and the covalent (Schiff's base) E-I species. Kinetic data analysis indicates Ki values of 175 and 11 microM for Ado-P2-Pl and Ado-P3-Pl, respectively. Thus, the ATP-binding domain can easily accommodate the pyridoxal moiety which is tethered to the polyphosphate chain. The phosphorylated ATP analogs employed in this study exhibit substantially tighter binding to phosphoribulokinase than does fluorosulfonyl-benzoyladenosine (Ki = 4.8 mM), which we have previously demonstrated to be useful in selectively modifying the ATP-binding domain (Krieger, T. J., and Miziorko, H. M. (1986) Biochemistry 25, 3496-3501; Krieger, T. J., Mende-Mueller, L. M., and Miziorko, H. M. (1987) Biochim. Biophys. Acta 915, 112-119). Although the adduct formed between oATP and enzyme was unsuitable for structural analysis, borohydride reduction of the Schiff's base formed between enzyme and Ado-P3-[3H]Pl produced a species useful for investigation by protein chemistry techniques. A radiolabeled tryptic peptide was prepared, isolated, and sequenced; the data indicate that lysine 68 is the residue modified by Ado-P3-[3H]Pl.     

Effect of culture conditions on production of 5-aminolevulinic acid by recombinant Escherichia coli

Aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (hemA gene). Then ALA is converted to Porphobilinogen (PBG) by the ALA dehydratase (hemB gene). For the overproduction of ALA, we used an Escherichia coli BL21(DE3) containing a hemA gene from Bradyrhzobium japonicum, which was created in our previous work. The effects of pH on the ALA synthase and ALA dehydratase were investigated. The ALA synthase and ALA dehydratase activities were dependent on the pH of the medium, with maximal activities occurring at pH 6.5 and 8.0 respectively. At pH 6.5, extracellular ALA reached 23 mM in a jar-fermenter. In addition, the effects of some nutritional factors, such as nitrogen source and the ratio of carbon to nitrogen (C/N) on the fermentative production of ALA were investigated. The highest ALA production was found with 8:1 of C/N ratio. Among various nitrogen sources, the tryptone might be a useful one for ALA production.