Double inhibition ofl-threonine dehydratase by aminothiols

The inhibition of highly purified rat liverl-threonine dehydratase (l-threonine hydro-lyase (deaminating), EC 4.2.1.16) by aminothiols (l-cysteine,d-cysteine, cysteamine) has been studied. Single inhibition experiments evaluated by Lineweaver-Burk and Dixon plots showed, in a given concentration range, partially (parabolic) competitive inhibitions, indicating two binding sites for each inhibitor. Double inhibition experiments revealed that the inhibition was antagonistic, the two inhibitors weakening each other's effect. Formation of EI₁ and EI₂ binary complexes, and ESI₁, ESI₂ and EI₁I₂ ternary complexes was demonstrated, while formation of the quaternary complex ESI₁I₂ was ruled out. It is assumed that one inhibitor-binding site coincides with the substrate-binding center while the second inhibitor-binding (allosteric, regulatory) site may comprise the pyridoxal-phosphate-binding SH group(s). The comparison between K_m and K_i values and the evaluation of intracellular concentrations ofl-threonine,l-cysteine and cysteamine suggest a possible physiological role of the inhibition.     

Double inhibition of L-threonine dehydratase by aminothiols

The inhibition of highly purified rat liver L-threonine dehydratase (L-threonine hydro-lyase (deaminating), EC 4.2.1.16) by aminothiols (L-cysteine, D-cysteine, cysteamine) has been studied. Single inhibition experiments evaluated by Lineweaver-Burk and Dixon plots showed, in a given concentration range, partially (parabolic) competitive inhibitions, indicating two binding sites for each inhibitor. Double inhibition experiments revealed that the inhibition was antagonistic, the two inhibitors weakening each other's effect. Formation of EI1 and EI2 binary complexes, and ESI1, ESI2 and EI1I2 ternary complexes was demonstrated, while formation of the quaternary complex ESI1I2 was ruled out. It is assumed that one inhibitor-binding site coincides with the substrate-binding center while the second inhibitor-binding (allosteric, regulatory) site may comprise the pyridoxal-phosphate-binding SH group(s). The comparison between Km and Ki values and the evaluation of intracellular concentrations of L-threonine, L-cysteine and cysteamine suggest a possible physiological role of the inhibition.     

Insect cell expression of recombinant imidazoleglycerolphosphate dehydratase of Arabidopsis and wheat and inhibition by triazole herbicides.

Imidazoleglycerolphosphate dehydratase (IGPD; EC 4.2.1.19), which is involved in the histidine biosynthetic pathway of Arabidopsis thaliana and wheat (Triticum aestivum), has been expressed in insect cells using the baculovirus expression vector system. N-terminal amino acid sequencing indicated that recombinant IGPDs (rIGPDs) were produced as mature forms via nonspecific proteolytic cleavages in the putative transit peptide region. The wheat rIGPD contained one Mn atom per subunit, and the Mn was involved in the assembly of the subunits to form active IGPDs. Protein-blotting analysis, using antibodies raised against the wheat rIGPD, indicated that IGPD was located in the chloroplasts of wheat. The rIGPDs of Arabidopsis and wheat, which were 86% identical in their primary structures deduced from the cDNAs, exhibited similar properties in terms of the molecular mass, pH optimum, and the Km for the substrate, imidazoleglycerolphosphate. However, the nonselective herbicides 3-amino-1,2,4-triazole and a newly synthesized triazole [(1R*, 3R*)-[3-hydroxy-3-(2H-[1,2,4]triazole-3-yl)-cyclohexyl]- phosphonic acid], inhibited Arabidopsis and wheat IGPDs in a mixed-type and a competitive manner, respectively.     

Temperature dependence of activity and inhibition by lead with 5-aminolevulinic acid dehydratase

5-Aminolevulinic acid dehydratase (EC 4.2.1.24) was purified 230-fold from human erythrocytes. The effects of temperature on various properties of the isolated enzyme were studied. The maximal activity is observed at 60 degrees C. The pH optimum is shifted from pH 7.3 at 5 degrees C to pH 6.6 at 55 degrees C. The Michaelis constant increases 5-fold from 5 to 55 degrees C. Inhibition by lead is more pronounced at elevated temperatures. This effect is in contradiction to the restoration of enzyme activity by heating hemolysate after inhibition by lead in vivo, which has been described in previous reports.     

The inhibition of rat liver threonine dehydratase by carbamoyl-phosphate. The formation of carbamoylpyridoxal 5'-phosphate

The effects exerted by carbamoyl phosphate (CP) and cyanate (KCNO) on rat liver L-threonine deaminase have been studied. The two compounds showed that same effects, inhibiting through a competitive mechanism both the holoenzyme and the dialyzed enzyme; inhibition was more evident for the latter. Ki values, both for L-threonine and pyridoxal 5'-phosphate (PLP), were lower for the apoenzyme and the inhibitors also affected the Km of the apoenzyme for PLP. The effects of CP and KCNO are mainly due to an interference in the association reaction apoenzyme + PLP in equilibrium holoenzyme This was clearly demonstrated by the fact that, when PLP was incubated with CP or KCNO, it failed to enhance the activity of the holoenzyme nor did it reactivate the resolved apoenzyme. Such interference of CP and KCNO in the L-threonine deaminase activity was mainly due to a specific mechanism, the formation of a new derivative of PLP. The reaction of PLP with either CP or KCNO occurred readily, at low concentrations, under physiological conditions. The new compound was identified as 3,4-dihydro-2H-pyrido[3,4-e]1,3-oxazin-2-one derivative by ultraviolet-visible spectra, elemental analysis, infrared, NMR and MS spectra. In this paper we formulate the hypothesis that this compound is involved in the regulation of the CP and PLP intracellular content and in the control of PLP dependent enzymes.     

Control of haem synthesis by feedback inhibition on human-erythrocyte δ-aminolaevulate dehydratase

The action of haem, haemoglobin and other haemoproteins on crude and purified δ-aminolaevulate dehydratase of human erythrocyte is described. The results show a feedback inhibition by haem of porphyrin synthesis at the level of erythrocyte δ-aminolaevulate dehydratase. Some kinetic characteristics of this inhibition are described.     

Metal constitution of metallothionein influences inhibition of delta-aminolaevulinic acid dehydratase (porphobilinogen synthase) by lead.

This study was undertaken to evaluate the effect of Zn and Cd pretreatment on the inhibition of delta-aminolaevulinic acid dehydratase (ALAD; porphobilinogen synthase, EC 4.2.1.24) by Pb. Male CD rats were pretreated with 200 mumol of Zn/kg s.c. (subcutaneously) or 18 mumol of Cd/kg s.c., 48 and 24 h before assay of ALAD. Pretreatment with Zn resulted in activation of hepatic and renal ALAD and attenuated the inhibition of this enzyme by Pb in vitro. Pretreatment with Cd increased hepatic ALAD activity, and the inhibitory effect of Pb on the hepatic enzyme was attenuated in this group. In contrast with the situation in liver, pretreatment with Cd did not affect the activity of renal ALAD and did not alter the inhibitory effect of Pb on the renal enzyme. The Pb IC50 (concentration causing half-maximal inhibition) values for hepatic and renal ALAD in Zn-pretreated rats and for hepatic ALAD in Cd-pretreated rats were increased above control, whereas the IC50 for renal ALAD in Cd-pretreated rats was unchanged. Cytosolic binding patterns for the three metals were assessed by gel-filtration chromatography and disclosed that 203Pb was co-eluted with Zn and Cd bound to liver and kidney Zn-thioneins and liver Cd,Zn-thionein, although minimal binding of 203Pb to kidney Cd,Zn-thionein was observed. Estimation of the molar ratio of metals bound revealed Cd/Zn ratios of 2 and 5 for Cd,Zn-thioneins from liver and kidney respectively. The inhibition of purified ALAD by Pb was also attenuated by addition of purified Zn-thioneins and Cd,Zn-thioneins from liver and kidney in the following order: liver Zn-thionein = kidney Zn-thionein greater than liver Cd,Zn-thionein much greater than kidney Cd,Zn-thionein. Thus liver and kidney Zn-thioneins and liver Cd,Zn-thionein with a low Cd/Zn ratio readily decrease the free pool of Pb available to interact with ALAD. These data also demonstrate that the capacity of metallothionein to alter the intracellular distribution of Pb and mediate the inhibition of ALAD by Pb is dependent on the tissue source and relative metal constitution of the metallothionein.     

Human erythrocyte delta-aminolevulinate dehydratase inhibition by monosaccharides is not mediated by oxidation of enzyme sulfhydryl groups

The heme pathway enzyme delta-aminolevulinate dehydratase is a good marker for oxidative stress and metal intoxication. This sulfhydryl enzyme is inhibited in such oxidative pathologies as lead, mercury and aluminum intoxication, exposure to selenium organic species and diabetes. Oxidative stress is a complicating factor in diabetes, inducing non-enzymatic glucose-mediated reactions that change protein structures and impair enzyme functions. We have studied the effects of high glucose, fructose and ribose concentrations on delta-ALA-D activity in vitro. These reducing sugars inhibited delta-ALA-D with efficacies in the order fructose=ribose>glucose. The possible mechanism of glucose inhibition was investigated using lysine, DTT, and t-butylamine. Oxidation of the enzyme's critical sulfhydryl groups was not involved because DTT had no effect. We concluded that high concentrations of reducing sugars or their autoxidation products inhibit delta-ALA-D by a mechanism not related to thiol oxidation. Also, we are not able to demonstrate that the formation of a Schiff base with the critical lysine residue of the enzyme is involved in the inhibition of delta-ALA-D by hexoses.     

Regulation of L-cystine transport in Salmonella typhimurium.

A kinetic analysis of L-cystine uptake in wild-type Salmonella typhimurium indicates the presence of at least two, and possibly three, separate transport systems. CTS-1 accounts for the majority of uptake at 20 muM L-cystine, with a Vmax of 9.5 nmol/min per mg and a Km of 2.0 muM; CTS-2 is a low-capacity, higher-affinity system with a Vmax of 0.22 nmol/min per mg and a Km of 0.05 muM; a third, nonsaturable process has been designated CTS-3. We find that wild-type CTS-1 levels are at least 11 times higher in sulfur-limited cells than in L-cystine-grown cells. Pleiotropic cysteine auxotrophs of the types cysE (lacking serine transacetylase) and cysB- (lacking a regulatory element of positive control) have very low levels of CTS-1 even when grown under conditions of sulfur limitation, which response is analogous to that previously observed for cysteine biosynthetic enzymes (N . M. Kredich, J. Biol. Chem. 246:3474-3484, 1971). CTS-1 is induced in cysE mutants by growth in the presence of O-acetyl-L-serine (the product of serine transacetylase), again paralleling the behavior of the cysteine biosynthetic pathway. Strain DW25, a prototrophic cysBc mutant, which is constitutive for cysteine biosynthesis, is also derepressed for CTS-1 when grown on L-cystine. Since CTS-1 is regulated by sulfur limitation, O-acetyl-L-serine, and the cysB gene product, the same three conditions controlling cysteine biosynthesis, we propose that this transport system is a part of the cysteine regulon.     

Hydrogen transfer in catalysis by adenosylcobalamin-dependent diol dehydratase.

Studies [bachovchin, W. W., et al. (1978) Biochemistry 17, 2218] of the mechanism of inactivation of adenosylcobalamin-dependent diol dehydratase have led to the development of a general method to describe the kinetics of a reaction pathway containing a reservoir of mobile hydrogen. Analysis by this method of catalytic rate measurements for mixtures of 1,2-propanediol and 1,1-dideuterio-1,2-propanediol supports a mechanism involving an intermediate with three equivalent hydrogens, in which hydrogen transfer from this intermediate to product is the major rate-contributing step. Other results using tritium as a trace label [essenberg, M. K., et al. (1971) J. Am. Chem. Soc. 93, 1242] are considered in light of these deuterium isotope studies.