Action Of INH Research Articles

Isoniazid-historical development, metabolism associated toxicity and a perspective on its pharmacological improvement.

Despite the extraordinary anti-tubercular activity of isoniazid (INH), the drug-induced hepatotoxicity and peripheral neuropathy pose a significant challenge to its wider clinical use. The primary cause of INH-induced hepatotoxicity is in vivo metabolism involving biotransformation on its terminal -NH2 group owing to its high nucleophilic nature. The human N-acetyltransferase-2 enzyme (NAT-2) exploits the reactivity of INH's terminal -NH2 functional group and inactivates it by transferring the acetyl group, which subsequently converts to toxic metabolites. This -NH2 group also tends to react with vital endogenous molecules such as pyridoxine, leading to their deficiency, a major cause of peripheral neuropathy. The elevation of liver functional markers is observed in 10%-20% of subjects on INH treatment. INH-induced risk of fatal hepatitis is about 0.05%-1%. The incidence of peripheral neuropathy is 2%-6.5%. In this review, we discuss the genesis and historical development of INH, and different reported mechanisms of action of INH. This is followed by a brief review of various clinical trials in chronological order, highlighting treatment-associated adverse events and their occurrence rates, including details such as geographical location, number of subjects, dosing concentration, and regimen used in these clinical studies. Further, we elaborated on various known metabolic transformations highlighting the involvement of the terminal -NH2 group of INH and corresponding host enzymes, the structure of different metabolites/conjugates, and their association with hepatotoxicity or neuritis. Post this deliberation, we propose a hydrolysable chemical derivatives-based approach as a way forward to restrict this metabolism.

Oral isoniazid causes oxidative stress, oocyte deterioration and infertility in mice

Isoniazid (INH), a synthetic first-line tuberculosis antibiotic, has been widely used in clinical treatment. It has been reported to cause toxic effects at multiple tissue sites and also increases the incidence of adverse pregnancy outcomes; but the mechanism of action of INH on the reproductive system of female mammals remains unclear. Here, we demonstrate that oral INH (40 mg/kg/day every other day for 28 days) severely affects oocyte maturation and fertilization, late blastocyst development and fertility. We found that INH could disrupt standard spindle assembly, chromosome arrangement, and actin filament dynamics, which compromised meiotic progression of mouse oocytes. INH treatment increased the level of reactive oxygen species (ROS) and activated the oxidative stress response pathway, Keap1-Nrf2. It also caused apoptosis of oocytes and mitochondrial dysfunction. Our findings demonstrate that oral INH reduces fertility and damages the mammalian reproductive system by altering cytoskeletal dynamics and Juno expression, inducing oxidative stress and apoptosis, and activating the Keap1-Nrf2 signaling pathway in mouse oocytes.

The investigation of the effect of NAD, H2O2 and weak magnetic field on the antibacterial mechanism of isoniazid (INH) that first line antibiotic against M. tuberculosis agent

Tuberculosis is an infectious disease, which is caused by the Mycobacterium tuberculosis complex. This disease leads to up to 1.3 million deaths out of more than eight million cases every year. A prodrug called isoniazid has been proven to be effective and widely used in the treatment of infections caused by tuberculosis. Despite its use for more than six decades clinically, the action mechanism of this prodrug is yet to be elucidated. INH action agains mycobacteria requires catalase−peroxidase (KatG) function, and IN-NAD adduct formation is catalyzed in vitro by M. tuberculosis KatG under a variety of conditions. Low-intensity EMF (Electromagnetic Field) has been used in the₂rapeutic practices in addition to its use in telecommunication systems and food protection. EMF is used in medicine and food industries especially for its bactericidal effects. In this study, we aimed to investigate the effects of weak magnetic field application and the addition of NAD and H₂O₂ on the action mechanism of isoniazid. We added H₂O₂ and NAD individually and together, to the different groups at varying concentrations. Also, one experimental group was exposed to a 5mT, 50Hz magnetic field for 4 to 5 hours per day (total of 45 hours in 10 days). The agar proportion method was used to evaluate the results. It was determined that the addition of 100 μM NAD and H₂O₂ together increased the effectiveness of isoniazid to some extent. However, the application of a weak magnetic field did not change the effectiveness of the drug.

Eosinophil peroxidase oxidizes isoniazid to form the active metabolite against M. tuberculosis, isoniazid-NAD+

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD+ adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD+ using purified EPO and eosinophils isolated from asthmatic donors. UV–Vis spectroscopy revealed INH oxidation by EPO led to a new product (λmax = 326 nm) in the presence of NAD+. This adduct was confirmed to be INH-NAD+ using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD+, was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD+ adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment.

Proteomic and morphological changes produced by subinhibitory concentration of isoniazid in Mycobacterium tuberculosis.

To study the proteomic and morphological changes in Mycobacterium tuberculosis H37Rv exposed to subinhibitory concentration of isoniazid (INH). The bacillus was exposed to ½ MIC of INH at 12, 24 and 48 h. The samples' cells were submitted to scanning electron microscopy. The proteins were separated by 2D gel electrophoresis and identified by MS. INH exposure was able to alter the format, the multiplication and causing a cell swelling in the bacillus. The major altered proteins were related to the virulence, detoxification, adaptation, intermediary metabolism and lipid metabolism. The protein and morphological changes in M. tuberculosis induced by ½ MIC INH were related to defense mechanism of the bacillus or the action of INH therein.

Modeling metabolic adjustment in Mycobacterium tuberculosis upon treatment with isoniazid

Complex biological systems exhibit a property of robustness at all levels of organization. Through different mechanisms, the system tries to sustain stress such as due to starvation or drug exposure. To explore whether reconfiguration of the metabolic networks is used as a means to achieve robustness, we have studied possible metabolic adjustments in Mtb upon exposure to isoniazid (INH), a front-line clinical drug. The redundancy in the genome of M. tuberculosis (Mtb) makes it an attractive system to explore if alternate routes of metabolism exist in the bacterium. While the mechanism of action of INH is well studied, its effect on the overall metabolism is not well characterized. Using flux balance analysis, inhibiting the fluxes flowing through the reactions catalyzed by Rv1484, the target of INH, significantly changes the overall flux profiles. At the pathway level, activation or inactivation of certain pathways distant from the target pathway, are seen. Metabolites such as NADPH are shown to reduce drastically, while fatty acids tend to accumulate. The overall biomass also decreases with increasing inhibition levels. Inhibition studies, pathway level clustering and comparison of the flux profiles with the gene expression data indicate the activation of folate metabolism, ubiquinone metabolism, and metabolism of certain amino acids. This analysis provides insights useful for target identification and designing strategies for combination therapy. Insights gained about the role of individual components of a system and their interactions will also provide a basis for reconstruction of whole systems through synthetic biology approaches. The online version of this article (doi:10.1007/s11693-011-9075-6) contains supplementary material, which is available to authorized users.

The Mechanism of Isoniazid Killing: Clarity Through the Scope of Genetics

Isoniazid (INH) is one of the most efficient drugs for the treatment of Mycobacterium tuberculosis infections. Despite its rather simple chemical structure, the mechanism by which INH kills M. tuberculosis is complex. A full understanding of the mechanisms of action of INH required the development of genetic tools in M. tuberculosis. Herein, we discuss the different hypotheses that have been used to describe INH action against M. tuberculosis over the past 50 years in terms of the pregenetic and genetic era. We also review the different mechanisms of INH resistance and propose what we think is the means by which INH kills M. tuberculosis.

Effects of Isoniazid on Viability, Cell Morphologies and Acid Fastness Properties of Mycobacterium avium NCTC 8559 during the Growth Cycle

Our previous study demonstrated that the effects of isoniazid (INH) on Mycobacterium tuberculosis at the cellular level varied according to the growth phases. In this study, the variations in the INH action on M. avium strain NCTC 8559 are reported. M. avium cells grown on Middlebrook 7H10 agar were harvested at different stages of their growth cycle, exposed to the minimum inhibitory concentration of INH, stained with acid-fast staining for morphological changes and acid fastness properties, and the number of colonies were evaluated for viability studies. The study demonstrated that M. avium NCTC 8559 cells at the initial and fragmentation stages of the growth cycle were most susceptible to INH.

Mechanisms of action of isoniazid

For decades after its introduction, the mechanisms of action of the front-line antituberculosis therapeutic agent isoniazid (INH) remained unclear. Recent developments have shown that peroxidative activation of isoniazid by the mycobacterial enzyme KatG generates reactive species that form adducts with NAD(+) and NADP(+) that are potent inhibitors of lipid and nucleic acid biosynthetic enzymes. A direct role for some isoniazid-derived reactive species, such as nitric oxide, in inhibiting mycobacterial metabolic enzymes has also been shown. The concerted effects of these activities - inhibition of cell wall lipid synthesis, depletion of nucleic acid pools and metabolic depression - drive the exquisite potency and selectivity of this agent. To understand INH action and resistance fully, a synthesis of knowledge is required from multiple separate lines of research - including molecular genetic approaches, in vitro biochemical studies and free radical chemistry - which is the intent of this review.

Understanding the action of INH on a highly INH-resistant Mycobacterium tuberculosis strain using Genechips

The availability of the complete sequence of Mycobacterium tuberculosis genome coupled with microarray technology has enabled a high-throughput approach to the pharmacogenomics of this organism. Isoniazid (INH) is a first-line drug for the treatment of tuberculosis and the microarray approach has generated new insight into the action of INH on a drug-susceptible strain. It has also shown that INH does not induce any significant change in gene expression when applied to a catalase-negative INH-resistant strain, which is expected because catalase activity is required to convert the prodrug INH to its active form. But it has yet to be determined how a partially resistant strain responds to INH. In this study, we explore the mechanism of INH against a highly INH-resistant strain, compare drug-induced gene-expression profiles between resistant and susceptible strains, and determine whether or not and how the resistant strain responds to INH at low and high concentrations. The global gene-expression profiles of the resistant strain in response to INH treatments were obtained using the Affymetrix oligonucleotide GeneChips. The results showed that the resistant strain did not exhibit the characteristic gene-expression signature of type II fatty acid synthase (FAS-II) inhibition when exposed to low-level INH, but it responded with that specific pattern under high-level INH, although the response profile was somewhat shrunken relative to that for a susceptible strain. We found that INH acted on the FAS-II pathway in both resistant and susceptible strains, and little evidence suggested that INH might kill resistant bacteria via other mechanisms. This suggests that there may be potential benefit of treating INH-resistant bacteria with INH at a level that is effective and safe.

Hydrogen Peroxide-Mediated Isoniazid Activation Catalyzed by Mycobacterium tuberculosis Catalase−Peroxidase (KatG) and Its S315T Mutant,

Inhibition of the enzyme Mycobacterium tuberculosis InhA (enoyl-acyl carrier protein reductase) due to formation of an isonicotinoyl-NAD adduct (IN-NAD) from isoniazid (INH) and nicotinamide adenine dinucleotide cofactor is considered central to the mode of action of INH, a first-line treatment for tuberculosis infection. INH action against mycobacteria requires catalase-peroxidase (KatG) function, and IN-NAD adduct formation is catalyzed in vitro by M. tuberculosis KatG under a variety of conditions, yet a physiologically relevant approach to the process has not emerged that allows scrutiny of the mechanism and the origins of INH resistance in the most prevalent drug-resistant strain bearing KatG[S315T]. In this report, we describe how hydrogen peroxide, delivered at very low concentrations to ferric KatG, leads to efficient inhibition of InhA due to formation of the IN-NAD adduct. The rate of adduct formation mediated by wild-type KatG was about 20-fold greater than by the isoniazid-resistant KatG[S315T] mutant under optimal conditions (H2O2 supplied along with NAD+ and INH). Slow adduct formation also occurs starting with NADH and INH, in the presence of KatG even in the absence of added peroxide, due to endogenous peroxide. The poor efficiency of the KatG[S315T] mutant can be enhanced merely by increasing the concentration of INH, consistent with this enzyme's reduced affinity for INH binding to the resting enzyme and the catalytically competent enzyme intermediate (Compound I). Origins of drug resistance in the KatG[S315T] mutant enzyme are analyzed at the structural level through examination of the three-dimensional X-ray crystal structure of the mutant enzyme.

Time-Action Profile of Inhaled Insulin in Comparison With Subcutaneously Injected Insulin Lispro and Regular Human Insulin

This study compares the time-action profile of inhaled insulin (INH; Exubera) with that of subcutaneously injected insulin lispro (ILP) or regular human insulin (RHI) in healthy volunteers. In this open-label, randomized, three-way, crossover study, 17 healthy male volunteers were given each of the following treatments in random order: INH (6 mg), ILP (18 units), or RHI (18 units). Glucose infusion rates and serum insulin concentrations were monitored over 10 h. INH had a faster onset of action than both RHI and ILP, as indicated by shorter time to early half-maximal effect (32 vs. 48 and 41 min, respectively; P < 0.001 for IHN vs. RHI and P < 0.05 for IHN vs. ILP). Time to maximal effect was comparable between INH and ILP (143 vs. 137 min; NS) but was shorter for INH than RHI (193 min; P < 0.01). The maximal metabolic effect of INH was comparable with RHI but lower than ILP (8.7 vs. 9.8 vs. 11.2 mg . kg(-1) . min(-1), respectively; P < 0.01 for INH vs. ILP). The duration of action of INH, indicated by time to late half-maximal effect (387 min), was longer than ILP (313 min; P < 0.01) and comparable to RHI (415 min; NS). Total glucodynamic effect after inhalation of INH was comparable to both ILP and RHI (NS). Relative bioefficacy of INH was 10% versus RHI and 11% versus ILP. No drug-related adverse events were observed. INH had a faster onset of action than RHI or ILP and a duration of action longer than ILP and comparable to RHI. These characteristics suggest that inhaled insulin is suitable for prandial insulin supplementation in patients with diabetes.

Requirements for nitric oxide generation from isoniazid activation in vitro and inhibition of mycobacterial respiration in vivo.

Isoniazid (INH), a front-line antituberculosis agent, is activated by mycobacterial catalase-peroxidase KatG, converting INH into bactericidal reactive species. Here we investigated the requirements and the pathway of nitric oxide (NO*) generation during oxidative activation of INH by Mycobacterium tuberculosis KatG in vitro. We also provide in vivo evidence that INH-derived NO* can inhibit key mycobacterial respiratory enzymes, which may contribute to the overall antimycobacterial action of INH.

Nitric oxide generated from isoniazid activation by KatG: source of nitric oxide and activity against Mycobacterium tuberculosis.

Isonicotinic acid hydrazide (INH) is a frontline antituberculosis agent. Once taken up by Mycobacterium tuberculosis, INH requires activation by the catalase-peroxidase KatG, converting INH from its prodrug form into a range of bactericidal reactive species. Here we used 15N-labeled INH together with electron paramagnetic resonance spin trapping techniques to demonstrate that nitric oxide (NO*) is generated from oxidation at the hydrazide nitrogens during the activation of INH by M. tuberculosis KatG. We also observed that a specific scavenger of NO* provided protection against the antimycobacterial activity of INH in bacterial culture. No significant increases in mycobacterial protein nitration were detected, suggesting that NOdot; and not peroxynitrite, a nitrating metabolite of NO*, is involved in antimycobacterial action. In conclusion, INH-derived NO* has biological activity, which directly contributes to the antimycobacterial action of INH.

The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance.

Isoniazid (INH), a frontline antitubercular drug, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. Here, we report that the INH-NAD adduct is a slow, tight-binding competitive inhibitor of InhA. Demonstration that the adduct binds to WT InhA by a two-step enzyme inhibition mechanism, with initial, weak binding (K(-1) = 16 +/- 11 nM) followed by slow conversion to a final inhibited complex (EI*) with overall Ki = 0.75 +/- 0.08 nM, reconciles existing contradictory values for the inhibitory potency of INH-NAD for InhA. The first order rate constant for conversion of the initial EI complex to EI* (k2 = 0.13 +/- 0.01 min(-1)) is similar to the maximum rate constant observed for InhA inhibition in reaction mixtures containing InhA, INH, NADH, and the INH-activating enzyme KatG (catalase/peroxidase from M. tuberculosis), consistent with an inhibition mechanism in which the adduct forms in solution rather than on the enzyme. Importantly, three mutations that correlate with INH resistance, I21V, I47T, and S94A, have little impact on the inhibition constants. Thus, drug resistance does not result simply from a reduction in affinity of INH-NAD for pure InhA. Instead, we hypothesize that protein-protein interactions within the FASII complex are critical to the mechanism of INH action. Finally, for M161V, an InhA mutation that correlates with resistance to the common biocide triclosan in Mycobacterium smegmatis, binding to form the initial EI complex is significantly weakened, explaining why this mutant inactivates more slowly than WT InhA when incubated with INH, NADH, and KatG.

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase InhA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of InhA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The InhA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of InhA inhibition. In addition, in vitro assays using purified InhA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited InhA but not KasA activity. Moreover, several thermosensitive InhA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of InhA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with InhA being the primary target of INH.

In Vitro and In Vivo Activities of Acylated Derivatives of Isoniazid Against Mycobacterium Tuberculosis

Enzymatic acylation of the antitubercular isoniazid (INH) by N-acetyl transferases reduces the therapeutic effectiveness of the drug. Because it represents a major metabolic pathway for INH in human beings, such acetylation has serious consequences for tuberculosis treatment regimens. Among patients in whom this process is efficient, the "rapid acetylators," the resultant chronic underdosing of INH may give rise to the development of resistance, as well as inadequate therapy. Not much work has been done previously to characterize the antitubercular properties of other N2-acylisoniazids. In order to address the fundamental issue of the activities of these acylated derivatives of INH, a number of such compounds 1a-f were chemically synthesized for investigation by a method providing good yield and purity. In experiments in vitro against Mycobacterium tuberculosis, these compounds displayed minimum inhibitory concentration (MIC) values between several fold and several hundred fold greater than that of INH itself, on a molar basis, with some of the more active compounds having higher calculated values of log P. Among these derivatives, compound 1b, closely homologous to the INH metabolite 1a, N2-acetylisoniazid, provided unexpected protection in tuberculosis-infected mice. The authors conclude that such close structural congeners of metabolites of INH may serve as significant leads in antitubercular drug discovery and in the exploration of the mode of action of INH.

In Vitro and In Vivo Activities of Acylated Derivatives of Isoniazid Against Mycobacterium Tuberculosis

Enzymatic acylation of the antitubercular isoniazid (INH) by N-acetyl transferases reduces the therapeutic effectiveness of the drug. Because it represents a major metabolic pathway for INH in human beings, such acetylation has serious consequences for tuberculosis treatment regimens. Among patients in whom this process is efficient, the "rapid acetylators," the resultant chronic underdosing of INH may give rise to the development of resistance, as well as inadequate therapy. Not much work has been done previously to characterize the antitubercular properties of other N2-acylisoniazids. In order to address the fundamental issue of the activities of these acylated derivatives of INH, a number of such compounds 1a-f were chemically synthesized for investigation by a method providing good yield and purity. In experiments in vitro against Mycobacterium tuberculosis, these compounds displayed minimum inhibitory concentration (MIC) values between several fold and several hundred fold greater than that of INH itself, on a molar basis, with some of the more active compounds having higher calculated values of log P. Among these derivatives, compound 1b, closely homologous to the INH metabolite 1a, N2-acetylisoniazid, provided unexpected protection in tuberculosis-infected mice. The authors conclude that such close structural congeners of metabolites of INH may serve as significant leads in antitubercular drug discovery and in the exploration of the mode of action of INH.

Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis.

The inhA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed inhA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either inhA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy inhA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing inhA plasmids uniformly exhibited 20-fold or greater increased resistance to INH and 10-fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH-susceptible M. tuberculosis strains. Using molecular beacons, quantified inhA and kasA mRNA levels showed that increased inhA mRNA levels corre--lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring inhA or kasA plasmids yielded the same conclusion: overexpressed inhA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, InhA is the primary target of action of INH and ETH in all three species.

Structural characterization of recombinant catalase-peroxidase from Mycobacterium tuberculosis.

It has long been observed that resistance to isoniazid (INH), the core compound used to treat tuberculosis, is often correlated with reduced levels of catalase activity [1,2]. It has also been confirmed that the presence of active catalaseperoxidase (CP), encoded by the katG gene, is necessary for INH sensitivity in M. tuberculosis [3]. Point mutations or deletions in katG can give rise to clinical isolates with increased levels of INH resistance [4]. The requirement for a katG gene product has been further supported by the observation that transformation of a plasmid harbouring this gene into INH-resistant M. smegmatis strains can restore INH sensitivity [5]. Although it is reasonable to assume that the presence of a functional catalase-peroxidase is required for INH sensitivity in M. tuberculosis, the mechanism of action of INH in the bacterium is not well defined. It has been shown that INH is capable of being oxidized by the katG gene product [6] however, the reactive intermediates responsible for the cellular effects of INH have yet to be identified. CP belongs to a family of bacterial enzymes which are believed to have evolved by a gene duplication [7] involving two modules to produce an 80 kDa subunit. Other catalaseperoxidase enzymes have been successfully purified from a variety of sources. They are generally multimers composed of identical subunits approximately 80 kDa in size. The CP enzymes from Escherichia coli [8] and from M. smegmatis 191 are tetrameric. CP can also be found in dimeric and monomeric form as well. The amino-terminal half of the CP enzyme family shows sequence homology with yeast cytochrome c peroxidase [lo]. The carboxy-terminal half may contain the catalase active site but lacks good sequence homology with known catalase structures. In order to develop a better understanding of the structural properties of CP, small-angle X-ray scattering (SAXS) experiments have been undertaken. Recombinant kutG-encoded M. tuberculosis CP (mtCP) was prepared by overproducing the enzyme in E. coli. The enzyme has been purified to homogeneity according to an established protocol [ l l , 121. mtCP was prepared in a range of concentrations in phosphate buffered saline (PBS). SAXS experiments have been carried out on beamline X33 of the EMBL storage ring DORIS of DESY (Deuthsches Elektronen Synchrotron, Hamburg, Germany). The results are shown in Figure 1 and 2. Over a protein concentration range between 2 and 35 mg/ml, CP appears to assemble as a dimer with an estimated molecular mass of 160 kDa. This result is consistent with gel filtration and native polyacrylamide gel electrophoresis studies which also demonstrated the presence of soluble, active protein in a dimeric state. Analysis of the experimental scattering curve (Figure 1) indicates a radius of gyration of 3.8 nm and an excluded volume of 306 nm3. This data has also been used to construct a molecular envelope (Figure 2) using a shape determination algorithm which utilises the two-fold symmetry of this molecule [ 131. This envelope suggests a head-to-head assembly of mtCP and is being used to guide the Biochemical Society Transactions (1 997) 25 S617