Abstract
The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is an approximately 97-kDa arginine-specific ADP-ribosyltransferase that is activated by proteolytic cleavage, thereby releasing the active 27-kDa enzyme (MTX(30-264)) and a 70-kDa C-terminal fragment (MTX(265-870)). In solution, the cleaved 70-kDa fragment is still a potent inhibitor of the ADP-ribosyltransferase activity of MTX. Here we studied the interaction of the 70-kDa fragment with the enzyme domain of MTX. Several C-terminal deletions of the 70-kDa fragment inhibited the enzymatic activity of MTX(30-264). However, the IC(50) values were about 2 orders of magnitude higher for the deletions than for the 70-kDa fragment. A peptide covering amino acid residues 265-285 of the holotoxin exhibited the same inhibitory potency as the C-terminal deletions of the 70-kDa fragment. MTX(265-285) contains several acidic residues, of which D273 and D275 were found to be essential for the inhibitory effect. Exchange of these residues in the 70-kDa fragment (MTX(265-870)) reduced its inhibitory potency. Kinetic analysis showed that the peptide MTX(265-285) had no effect on the V(max) of MTX(30-264) but increased the K(m) for NAD. By contrast, the 70-kDa fragment deleted of residues Ile265 through Asn285 inhibited the enzyme activity of MTX(30-264) mainly by decreasing the V(max) of the enzyme. A second binding site for interaction of MTX(265-870) with MTX(30-264) was localized to the C-terminus within the region of residues 750-870. The data support a two-site binding model for inhibition of the ADP-ribosyltransferase activity of MTX(30-264) by the 70-kDa fragment MTX(265-870) with an interaction of amino acid residues 265-285 at the active site and an allosteric inhibition by the C-terminal part of the 70-kDa fragment.
Published Version
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