The purification and properties of peroxidase in Mycobacterium tuberculosis H37Rv and its possible role in the mechanism of action of isonicotinic acid hydrazide.
Peroxidase from Mycobacterium tuberculosis H37Rv was purified to homogeneity. The homogeneous protein exhibits catalase and Y (Youatt's)-enzyme activities in addition to peroxidase activity. Further confirmation that the three activities are due to a single enzyme was accomplished by other criteria, such as differential thermal inactivation, sensitivity to different inhibitors, and co-purification. The Y enzyme (peroxidase) was separated from NADase (NAD+ glycohydrolase) inhibitor by gel filtration on Sephadex G-200. The molecular weights of peroxidase and NADase inhibitor, as determined by gel filtration, are 240000 and 98000 respectively. The Y enzyme shows two Km values for both isoniazid (isonicotinic acid hydrazide) and NAD at low and high concentrations. Analysis of the data by Hill plots revealed that the enzyme has one binding site at lower substrate concentrations and more than one at higher substrate concentration. The enzyme contains 6g-atoms of iron/mol. Highly purified preparations of peroxidases from different sources catalyse the Y-enzyme reaction, suggesting that the nature of the reaction may be a peroxidatic oxidation of isoniazid. Moreover, the Y-enzyme reaction is enhanced by O2. Isoniazid-resistant mutants do not exhibit Y-enzyme, peroxidase or catalase activities, and do not take up isoniazid. The Y-enzyme reaction is therefore implicated in the uptake of the drug.
D002374:Catalase / Q000302:isolation & purification / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007538:Isoniazid / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D010544:Peroxidases / Q000302:isolation & purification / Q000378:metabolism*; D013045:Species Specificity; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D009243:NAD; D007501:Iron; D006735:Horseradish Peroxidase; D010544:Peroxidases; D002374:Catalase; D007538:Isoniazid