Substrate Specificities of Peroxidase Isozymes in the Developing Pea Seedling

The peroxidase activities of developing pea seedlings were determinedwith several substrates including three phenolic compounds,eugenol, caffeic acid and ferulic acid, which are possible precursorsin the biosynthesis of lignin. Column chromatography of thereaction products of peroxidase with caffeic and ferulic acidsindicates the formation of larger molecular weight complexesof these substrates. The peroxidase isozymes of peas were shownto be heterogenous both in molecular weight and in substratespecificity. Apparent K_m determinations of two isolated isozymesindicate differences in affinities for various substrates. Starchgel zymograms with two different substrates also indicate largedifferences in staining intensities of the different isozymes.The observed pattern of changes in peroxidase level in the developingpea seedling differed according to substrate. For example, whencaffeic acid is the hydrogen donor, a large increase in activitywas observed in the 6th to 8th day of germination. This peakof activity was not observed with other substrates. Pisum sativum, peroxidase isozymes, substrate specificity     

Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities

Caseinolytic (Clp) proteases are widespread energy-dependent proteases; the functional ATP-dependent protease is comprised of multimers of proteolytic and regulatory subunits. Mycobacterium tuberculosis has two ClpP proteolytic subunits (ClpP1 and ClpP2), with both being essential for growth in vitro. ClpP1 and clpP2 are arranged in an apparent operon; we demonstrated that the two genes are co-expressed under normal growth conditions. We identified a single promoter region for the clpP1P2 operon; no promoter was detected upstream of clpP2 demonstrating that independent expression of clpP1 and clpP2 was highly unlikely. Promoter activity was not induced by heat shock or oxidative stress. We identified a regulatory region upstream of the promoter with a consensus sequence matching the ClgR regulator motif; we determined the limits of the region by mutagenesis and confirmed that positive regulation of the promoter occurs in M. tuberculosis. We developed a reporter system to monitor ClpP1 and ClpP2 enzymatic activities based on LacZ incorporating ssrAtag sequences. We showed that whilst both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, ClpP2 (but not ClpP1) degrades untagged proteins. Our data suggest that the two proteolytic subunits display different substrate specificities and therefore have different, but overlapping roles in M. tuberculosis.