Nevertheless, pathway signaling the enzyme retained 85% of its activity over a broad tem perature range 30 50 C suggesting stability and absence of regulation depending on the T. cruzi host. In contrast, rLAPTc exhibits a distinct activity pro file at different temperatures, specific activity measured at 37 C corresponded to only 25% of the recorded maxi mal activity observed at 60 C. These data indicate that the native enzyme is mesophilic, whereas its recombinant form produced in E. coli is thermophi lic. To study the thermostability of LAPTc, hydrolysis of Leu AMC by native and recombinant forms of the enzyme was assayed at 37 or 60 C, respectively, after preincubation at different temperatures for either 15 or 240 min. Under these experimental conditions, the enzymatic activity of LAPTc was not significantly modified after preincubation at 37 C for 240 min.

How ever, preincubation at higher temperatures resulted in significant loss of enzymatic activity. rLAPTc was shown to be more stable than its native form, which correlates well with its higher optimal temperature of activity. The Michaelis Menten constant and maximal velocity of LAPTc were determined according to the hyperbolic regression method. The endogenous enzyme has a Km value of 12. 0 0. 8 uM Leu AMC and its calculated catalytic constant and catalytic effi ciency are 12. 47 1. 2 S 1 and 1. 04 0. 09 uM 1 rLAPTc are 185. 9 17. 0 uM, 34. 84 2. 9 S 1 and 0. 19 0. 01 uM 1. S 1, in that order. These results show that native and recombinant LAPTc exhibit different kinetic parameters.

LAPTc retains its oligomeric structure after losing activity We asked whether the temperature dependent enzy matic inactivation of LAPTc was due to monomeriza tion of the oligomer. This question was addressed by incubating LAPTc for 15 min at different temperatures, followed by SDS PAGE analysis. Although its enzymatic activity was almost completely lost at 60 C, the pepti dase fully retained its oligomeric form upon preincuba tion up to 80 C. Complete disassembly of the oligomer was achieved after boiling the sample, since LAPTc migrated as a single 55 kDa band in the gel. These data indicate that LAPTc keeps its oligomeric form after temperature induced inactivation. On the other hand, rLAPTc monomerization as a function of temperature correlates well with its loss of activity.

LAPTc is a metalloaminopeptidase The enzymatic activity of LAPTc on Leu AMC was completely inhibited by 100 uM bestatin, while 250 uM 1,10 phenanthroline and 10 mM EDTA inactivated 83 and 45% of the peptidase activity, respectively. Brefeldin_A LAPTc hydrolytic activity was not sensitive to PMSF, TLCK, E 64, leupeptin or pepstatin A. The activity of the enzyme previously inactivated by EDTA or 1,10 phenanthroline was potentiated by 0. 4 mM Mn2 or Ca2 polyclonal antibodies raised against the purified enzyme.