Positional isotope exchange analysis of the Mycobacterium smegmatis cysteine ligase (MshC)

MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins, which is an intermediate in the biosynthetic pathway of mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (MSH or AcCys-GlcN-Ins). MSH is produced by Mycobacterium tuberculosis, members of the Actinomycetes family, to maintain an intracellular reducing environment and protect against oxidative and antibiotic induced stress. The biosynthesis of MSH is essential for cell growth, and therefore, the MSH biosynthetic enzymes present potential targets for inhibitor design. The formation of kinetically competent adenylated intermediates was suggested by the observation of positional isotope exchange (PIX) reaction using [betagamma-(18)O6]-ATP in the presence of cysteine. The PIX rate depends on the presence of cysteine and increases with concentrations of cysteine. The loss of PIX activity upon the addition of small concentrations of pyrophosphatase suggests that the PP(i) is free to dissociate from the active site of cysteine ligase into the bulk solution. The PIX activity is also eliminated at high concentrations of GlcN-Ins, consistent with the mechanism in which GlcN-Ins binds after cysteine-adenylate formation. This PIX analysis confirms that MshC catalyzes the formation of a kinetically competent cysteinyl-adenylate intermediate after the addition of ATP and cysteine.     

Mycothiol import by Mycobacterium smegmatis and function as a resource for metabolic precursors and energy production

Mycothiol ([MSH] AcCys-GlcN-Ins, where Ac is acetyl) is the major thiol produced by Mycobacterium smegmatis and other actinomycetes. Mutants deficient in MshA (strain 49) or MshC (transposon mutant Tn1) of MSH biosynthesis produce no MSH. However, when stationary phase cultures of these mutants were incubated in medium containing MSH, they actively transported it to generate cellular levels of MSH comparable to or greater than the normal content of the wild-type strain. When these MSH-loaded mutants were transferred to MSH-free preconditioned medium, the cellular MSH was catabolized to generate GlcN-Ins and AcCys. The latter was rapidly converted to Cys by a high deacetylase activity assayed in extracts. The Cys could be converted to pyruvate by a cysteine desulfhydrase or used to regenerate MSH in cells with active MshC. Using MSH labeled with [U-(14)C]cysteine or with [6-(3)H]GlcN, it was shown that these residues are catabolized to generate radiolabeled products that are ultimately lost from the cell, indicating extensive catabolism via the glycolytic and Krebs cycle pathways. These findings, coupled with the fact the myo-inositol can serve as a sole carbon source for growth of M. smegmatis, indicate that MSH functions not only as a protective cofactor but also as a reservoir of readily available biosynthetic precursors and energy-generating metabolites potentially important under stress conditions. The half-life of MSH was determined in stationary phase cells to be approximately 50 h in strains with active MshC and 16 +/- 3 h in the MshC-deficient mutant, suggesting that MSH biosynthesis may be a suitable target for drugs to treat dormant tuberculosis.     

Evaluation of NTF1836 as an inhibitor of the mycothiol biosynthetic enzyme MshC in growing and non-replicating Mycobacterium tuberculosis

The mycothiol biosynthesis enzyme MshC catalyzes the ligation of cysteine with the pseudodisaccharide GlcN-Ins and has been identified as an essential enzyme in Mycobacterium tuberculosis. We now report on the development of NTF1836 as a micromolar inhibitor of MshC. Using commercial libraries, we conducted preliminary structure-activity relationship (SAR) studies on NTF1836. Based on this data, NTF1836 and five structurally related compounds showed similar activity towards clinical strains of M. tuberculosis. A gram scale synthesis was developed to provide ample material for biological studies. Using this material, we determined that inhibition of M. tuberculosis growth by NTF1836 was accompanied by a fall in mycothiol and an increase in GlcN-Ins consistent with the targeting of MshC. We also determined that NTF1836 kills non-replicating M. tuberculosis in the carbon starvation model of latency.     

ATP-dependent L-cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase,mycothiol biosynthesis enzyme MshC,is related to class I cysteinyl-tRNA synthetases

Mycothiol is a novel thiol produced only by actinomycetes and is the major low molecular weight thiol in mycobacteria. The mycothiol biosynthetic pathway has been postulated to involve ATP-dependent ligation of L-cysteine (Cys) with 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside; GlcN-Ins) catalyzed by MshC to produce Cys-GlcN-Ins. The ligase activity was purified approximately 2400-fold from Mycobacterium smegmatis and two proteins of slightly different M(r) approximately 47000 were identified with MshC activity. The N-terminal sequence of the smaller protein revealed that it was coded by a gene in the databases for M. smegmatis and M. tuberculosis previously designated as cysS2. The larger protein was coded by the same gene in M. smegmatis but included an eight amino acid N-terminal extension involving a different start codon. The ligase was found to have K(m) values of 40 +/- 3 and 72 +/- 9 microM for Cys and GlcN-Ins, respectively. The cysS2 gene was thought to encode a second cysteinyl-tRNA synthetase in addition to cysS but the present results indicate that cysS2 is actually the mshC gene encoding ATP-dependent Cys:GlcN-Ins ligase.     

A coupled spectrophotometric assay for l-cysteine:1-D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase and its application for inhibitor screening

Most actinomycetes, including Mycobacterium tuberculosis, do not produce glutathione but make an alternative thiol, mycothiol, which has functions similar to those of glutathione. A key step in mycothiol biosynthesis is the ATP-dependent ligation of Cys to GlcN-Ins catalyzed by MshC to produce Cys-GlcN-Ins, AMP, and PP(i). MshC is essential for growth of M. tuberculosis and is therefore a potential target for drugs directed against tuberculosis. A coupled-enzyme assay for MshC was developed using pyrophosphatase to convert pyrophosphate to phosphate and spectrophotometric detection of the latter via the phosphomolybdate complex with malachite green. The assay was readily adapted for use in a 96-well microtiter plate format. A secondary high-performance liquid chromatography assay measuring Cys-GlcN-Ins production was used to validate potential hits. Preliminary testing on a library of 2,024 compounds predicted to inhibit ATP-dependent enzymes identified many promiscuous and pyrophosphatase inhibitors of MshC and a single validated inhibitor with IC(50) approximately 100 microM.     

Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase.

Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast and plants. Pantothenate synthetase from Mycobacterium tuberculosis was expressed in E. coli, purified to homogeneity, and found to be a homodimer with a subunit molecular mass of 33 kDa. Initial velocity, product, and dead-end inhibition studies showed the kinetic mechanism of pantothenate synthetase to be Bi Uni Uni Bi Ping Pong, with ATP binding followed by D-pantoate binding, release of PP(i), binding of beta-alanine, followed by the release of pantothenate and AMP. Michaelis constants were 0.13, 0.8, and 2.6 mM for D-pantoate, beta-alanine, and ATP, respectively, and the turnover number, k(cat), was 3.4 s(-1). The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by (31)P NMR spectroscopy of [(18)O]AMP produced from (18)O transfer using [carboxyl-(18)O]pantoate. Single-turnover reactions for the formation of pyrophosphate and pantothenate were determined using rapid quench techniques, and indicated that the two half-reactions occurred with maximum rates of 1.3 +/- 0.3 and 2.6 +/- 0.3 s(-)(1), respectively, consistent with pantoyl adenylate being a kinetically competent intermediate in the pantothenate synthetase reaction. These data also suggest that both half-reactions are partially rate-limiting. Reverse isotope exchange of [(14)C]-beta-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products.     

Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics

Mycothiol, MSH or 1D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, is an unusual conjugate of N-acetylcysteine (AcCys) with 1D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins), and is the major low-molecular-mass thiol in mycobacteria. Mycothiol has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Because of these activities, MSH is a candidate for protecting Mycobacterium tuberculosis from inactivation by the host during infections as well as for resisting antituberculosis drugs. In order to define the protective role of MSH for M. tuberculosis, we have constructed an M. tuberculosis mutant in Rv1170, one of the candidate MSH biosynthetic genes. During exponential growth, the Rv1170 mutant bacteria produced approximately 20% of wild-type levels of MSH. Levels of the Rv1170 substrate, GlcNAc-Ins, were elevated, whereas those of the product, GlcN-Ins, were reduced. This establishes that the Rv1170 gene encodes for the major GlcNAc-Ins deacetylase activity (termed MshB) in the MSH biosynthetic pathway of M. tuberculosis. The Rv1170 mutant grew poorly on agar media lacking catalase and oleic acid, and had heightened sensitivities to the toxic oxidant cumene hydroperoxide and to the antibiotic rifampin. In addition, the mutant was more resistant to isoniazid, suggesting a role for MSH in activation of this prodrug. These data indicate that MSH contributes to the protection of M. tuberculosis from oxidants and influences resistance to two first-line antituberculosis drugs.     

Mycothiol biochemistry

Mycothiol (MSH) is a novel thiol comprised of N-acetylcysteine amide-linked to GlcN-alpha(1-1)-Ins. It is the major thiol in most actinomycetes and is produced at millimolar levels in mycobacteria and streptomycetes. MSH biosynthesis occurs by linkage of GlcNAc to Ins, deacetylation to GlcN-Ins, ligation of the latter to L-cysteine, and transacetylation of the cysteinyl residue by CoASAc to produce MSH. The genes encoding the respective enzymes have been designated mshA, mshB, mshC, and mshD; all but mshA have been identified. Mycobacterium smegmatis mutants deficient in mshA, mshC, and mshD have been characterized. MSH plays a significant role in the detoxification of thiol-reactive substances, including formaldehyde, various electrophiles, and antibiotics. Mycothiol S-conjugates derived from electrophiles and antibiotics are cleaved by mycothiol S-conjugate amidase to release GlcN-Ins, used to resynthesize MSH, and a mercapturic acid which is excreted from the cell. A mycothiol-disulfide-selective reductase has been identified and likely helps to maintain cellular MSH in the reduced state. Mycothiol biochemistry has characteristics similar to those of glutathione but also has a variety of unique features.     

Kinetic mechanism of AKT/PKB enzyme family

AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.     

A two-site kinetic mechanism for ATP binding and hydrolysis by E. coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide.

Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10 % (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (kcat=17(+/-2) s-1; KM=3 microM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-Pi) is preceded by a slow rate-limiting isomerization ( approximately 0.2 s-1). However, product (ADP or Pi) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses. The binding of the first ATP occurs by a two-step mechanism in which binding (k+1=1.5(+/-0.2)x10(7) M-1 s-1, k-1=29(+/-2) s-1) is followed by a protein conformational change (k+2=23(+/-3) s-1), monitored by an enhancement of Trp fluorescence. The second Trp fluorescence quenching phase is associated with binding of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-Pi). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.     

Studies on the mechanism of the glutamine-dependent reaction catalyzed by asparagine synthetase from mouse pancreas

Initial velocity and product inhibition studies were conducted with the glutamine-dependent reaction of asparagine synthetase from mouse pancreas. Double reciprocal plots of glutamine versus either aspartate or ATP were parallel, while aspartate versus ATP gave intersecting patterns. These patterns are indicative of a hybrid ping-pong mechanism consisting of a glutaminase partial reaction and a sequential catalysis involving aspartate and ATP. Inhibition patterns of the four products, glutamate, AMP, PPi, and asparagine, versus each of the three substrates are consistent with a hybrid Uni Uni Bi Ter Ping Pong Theorell-Chance mechanism where the glutaminase reaction occurs first and aspartate binds to the enzyme before ATP in the sequential segment. PPi is the first product released in the Theorell-Chance reaction, which is followed by the ordered release of AMP and asparagine. Product inhibition patterns also indicate the formation of E . NH3 . Asn and E . NH3 . Asp . AMP abortive complexes. Although an amide site (for glutamine and asparagine), presumably responsible for the glutaminase reaction, an acid site (for glutamate and aspartate), and a nucleotide site are involved in the overall catalysis, the "two-site" ping-pong mechanism is incompatible with the experimentally observed product inhibition patterns.     

Kinetic studies of bovine liver fructose-1,6-bisphosphatase.

Initial rate kinetic studies with bovine liver fructose-1,6-bisphosphatase were carried out in both directions of the reaction to determine the sequence of product release from the enzyme. Product inhibition by fructose-6-P was found to be S-linear, I-linear noncompetitive relative to fructose-1,6-bisphosphate, whereas inorganic orthophosphate was determined to be linear competitive with respect to the substrate. The kinetics of the reverse reaction were studied by coupling the phosphatase reaction to the aldolase, triosephosphate isomerase, and glycerolphosphate dehydrogenase reactions. The kinetic results were found to be in harmony with the Uni Bi ordered and random sequential mechanisms as well as a Uni Bi ping-pong mechanism. The nomenclature is that of Cleland (Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104-137). However, nonkinetic considerations, when taken together with the kinetic results, suggest that the steady state ordered Uni Bi mechanism is the most likely possibility. There is evidence that isomerization of the binary complex of enzyme and phosphate occurs in the kinetic mechanism. Although magnesium is required for the reverse reaction, there is no evidence to suggest that the enzyme discriminates between the magnesium-associated or divalent cation-free forms of the substrates.     

Kinetic Basis for the Conjugation of Auxin by a GH3 Family Indole-acetic Acid-Amido Synthetase

The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg²⁺ or Mn²⁺ for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4–9-fold reductions in k_cat/K_m relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.     

Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism

The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.     

Kinetic mechanism of fully activated S6K1 protein kinase

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His(6)-S6K1alphaII(DeltaAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (K(d)(ATP)=5-6 microM) was followed by low affinity binding of Tide (K(d)(Tide)=180 microM), and values of K(m)(ATP)=5-6 microM and K(m)(Tide)=4-5 microM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.     

Kinetic mechanism of arginyl-tRNA synthetase from human placenta

Like arginyl-tRNA synthetases from other organisms, human placental arginyl-tRNA synthetase catalyzes the arginine-dependent ATP-PPi exchange reaction only in the presence of tRNA. We have investigated the order of substrate addition and product release of this human enzyme in the tRNA aminoacylation reaction by using initial velocity experiments and dead-end product inhibition studies. The kinetic patterns obtained are consistent with a random Ter Ter sequential mechanism, instead of the common Bi Uni Uni Bi ping-pong mechanism for all other human aminoacyl-tRNA synthetases so far investigated in this respect.     

Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23)

The emergence of drug-resistant strains of Mycobacterium tuberculosis, the major causative agent of tuberculosis (TB), and the deadly HIV-TB co-infection have led to an urgent need for the development of new anti-TB drugs. The histidine biosynthetic pathway is present in bacteria, archaebacteria, lower eukaryotes and plants, but is absent in mammals. Disruption of the hisD gene has been shown to be essential for M. tuberculosis survival. Here we present cloning, expression and purification of recombinant hisD-encoded histidinol dehydrogenase (MtHisD). N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtHisD. Analytical gel filtration, metal requirement analysis, steady-state kinetics and isothermal titration calorimetry data showed that homodimeric MtHisD is a metalloprotein that follows a Bi Uni Uni Bi Ping-Pong mechanism. pH-rate profiles and a three-dimensional model of MtHisD allowed proposal of amino acid residues involved in either catalysis or substrate(s) binding.     

Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside and mycothiol biosynthesis

Mycothiol (MSH) is the major low molecular weight thiol in mycobacteria. Two chemical mutants with low MSH and one with no MSH (strain 49) were produced in Mycobacterium smegmatis mc2155 to assess the role of MSH in mycobacteria. Strain 49 was shown to not produce 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside (GlcN-Ins), an intermediate in MSH biosynthesis. Relative to the parent strain, mutant 49 formed colonies more slowly on solid media and was more sensitive to H2O2 and rifampin, but less sensitive to isoniazid. Complementation of mutant 49 with DNA from M. tuberculosis H37Rv partially restored production of GlcN-Ins and MSH, and resistance to H2O2, but largely restored colony growth rate and sensitivity to rifampin and isoniazid. The results indicate that MSH and GlcN-Ins are not essential for in vitro survival of mycobacteria but may play significant roles in determining the sensitivity of mycobacteria to environmental toxins.     

Reverse direction substrate kinetics and inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for ATP phosphoribosyltransferase were determined in the direction reverse to the biosynthetic reaction and are consistent with a sequential kinetic mechanism. Histidine inhibited the reverse reaction cooperatively and completely. Product and alternate product inhibition studies were conducted to elucidate binding order. The alternate product β,γ-methylene ATP was competitive with respect to N¹-phosphoribosyl-ATP and noncompetitive with respect to pyrophosphate. Phosphoribosylpyrophosphate was noncompetitive with respect to both substrates. These data and those of the biosynthetic direction reaction are in satisfactory quantitative agreement with the ordered Bi-Bi kinetic mechanism with ATP or phosphoribosyl-ATP binding to free enzyme.