Figure 2

Mutations of active site residues affect enzymatic activity of Mt MetAPs. (A) Sequence alignment of the two mycobacterial methionine aminopeptidases was performed using Clustal X. Gaps in the sequences were introduced for optimum alignment. Asterisk and dots denote, identical and similar amino acids, respectively. Residues highlighted with black represent the 40 amino acid long N-terminal extension present in Mt MetAP1c and those with gray are the mutated amino acids in the two Mt MetAPs. (B) Structural alignment of Mt MetAP1a (green) with respect to Mt MetAP1c (pink). Residues in green (sticks) represent Mt MetAP1a active site residues (His-88, His-193, Glu-219) and residues in pink (sticks) depict Mt MetAP1c active site residues (His212, His-114, Glu-238). Residue in blue is Tyr-183 (Mt MetAP1a) and in hot pink is Phe-202 (Mt MetAP1c). (C) Methionine aminopeptidase activity of different point mutants. Enzyme activity for the indicated amount of wild-type and purified variants of two Mt MetAP1s was monitored using 4 mM of substrate, Met-Gly-Met-Met (Mt MetAP1a) and Met-Ala-Ser (Mt MetAP1c). Insets, Western blot of mutant proteins using anti-His antibody, far-UV and near-UV CD spectra.