Comparison of the Catalytic Activities of Ricin A-Chain, Maize rproRIP1, MaizeRIP1, and Two Maize rproRIP1 Deletion Mutants Interacting with an RNA 10-mer GAGA Tetraloop.

Abstract

Ribosome-inactivating proteins (RIPs) catalytically depurinate a conserved adenine in the ribosomal alpha-sarcin domain, and it is believed that the local structure surrounding the target adenine is (at least transiently) an RNA GAGA tetraloop with the first A in the GAGA sequence being the target adenine. Studies of the catalytic activity of ricin A-chain interacting with ribosomes and with RNA GAGA tetraloops, as well as the catalytic activities of a set of related maize RIPs interacting with ribosomes have been reported in the literature. The purpose of this research project was to extend our understanding of maize RIP1 catalytic activity by measuring and comparing the catalytic activities of ricin A-chain and a set of related maize RIPs interacting with an RNA 10-mer GAGA tetraloop (denoted by A-10) over a range of pH values from pH 3.0 to 6.0.

Timecourse experiments of A-10/enzyme reactions were used to measure catalytic activity by quantitative HPLC detection of the adenine released during a succession of elapsed time intervals as the depurination reaction proceeded. The form of the timecourse data did not conform sufficiently to the integrated Michaelis-Menten equation to permit the use of nonlinear curve fitting to characterize the results in terms of the Michaelis-Menten parameters K[subscript M] and k[subscript cat]. Thus, the comparison of the activities of ricin A-chain and the various maize RIP variants both among themselves and as a function of pH, although definitive, is semiquantitative. For one of the enzymes, maizeRIP1, initial velocity experiments at high substrate concentrations yielded an approximate V[subscript max] directly, which was then used to treat V[subscript max] as a constant rather than a variable in fitting the data to the integrated Michaelis-Menten equation, and corresponding values for K[subscript max] were obtained. However, these substrate concentrations were not sufficiently high to yield V[subscript max] values for the other enzymes.

One unexpected result, with important ramifications concerning the conformation of the maize proRIP1, was evidence that maize rproRIP1, which is a zymogen (inactive precursor) with respect to ribosomes, may be catalytically active with respect to the small RNA tetraloop A-10.