Restrictocin is a 149 amino acid ribonucleolytic toxin produced by the fungus Aspergillus. which specifically cleaves a single phosphodiester bond within 28S rRNA resulting in a potent inhibition of protein synthesis in eukaryotic cells. Restrictocin has 12 prolines out of which three at positions 48, 112. and 126 are cis. Prolines at position 112. 181. and 126 were individually mutated to alanine to investigate their role in the catalytic and membrane interaction activity of restrictocin. All mutants were expressed in Escherichia coli, and recombinant proteins purified to homogeneity. Mutation of P112 resulted in a remarkable 50- and 100-fold reduction. respectively, in the ribonucleolytic and cytotoxic activities of restrictocin, whereas the interaction of P112A with phospholipid membranes increased. Mutants P118A and P126A exhibited 3-5-fold decreased ribonucleolytic and cytotoxic activities, however, their membrane interaction activity was marginally reduced compared to restrictocin. The study demonstrates that P112 is absolutely essential to maintain the functionally active conformation of restrictocin. Also, prolines 112, 118, and 126 do not appear to be directly involved in the membrane interaction activity of restrictocin. (C) 2002 Elsevier Science (USA). All rights reserved.