Onconase is the smallest member of pancreatic ribonuclease superfamily: it has 104 residues against 124 of RNase A and shares only 30 % of sequence identity with RNase A. Notwithstanding, the X-ray structure of onconase at 1.9 Å resolution [1] indicates, unequivocally, that the protein possesses a topology closely similar to that of RNase A, with the only significant differences located in loop regions and at the C-terminus, where onconase has a disulphide bond, Cys87-Cys104, which is found only in frog ribonucleases. Onconase has a catalytic activity quite low compared to that of RNase A, but it possesses a specific antitumor activity [2], and is currently undergoing phase III human clinical trials in the U.S.A. Catalytic activity is necessary for the antitumor action since carboxymethylation of the two histidines in the active site, His10 and His97, respectively, removes both activities. An efficient procedure, to obtain recombinant onconase and some mutants with high yield from a secretion system in E.coli, has been developed in our labs.

Differential scanning calorimetry, DSC, measurements are performed to characterize the thermal stability of onconase and its mutant M23L in a wide pH range. Experimental data indicate that onconase has an exceptional thermal stability, resembling thermophilic proteins, even though it is a mesophilic enzyme. Indeed, its denaturation temperature is close to 90 °C around pH 6.0 and is still close to 70 °C at pH 2.0. In this respect, a crucial role seems to be played by the disulfide bond at the C-terminus. On the other hand, the values of denaturation temperature for M23L-onconase are lower than those of parent enzyme by about 6 Celsius degrees. GuHCl-induced denaturation of the two proteins, probed by both circular dichroism and fluorescence measurements, lead to very similar results for the thermodynamic stability of onconase and it mutant M23L.

Methionine 23 is the last residue in the second helix of onconase (this helix is very short, ranging from Cys19 up to Met23, and distorted, being a mixture of a- and 310-helices), and is packed against the first of the two b-sheets constituting the characteristic V-shaped motif of the ribonuclease fold. A molecular modelling study indicates that leucine, when introduced in position 23 of onconase, can adopt a more compact conformation than methionine, creating a small cavity at the interface between the two secondary structure elements. This non-optimal packing between the helix and the b-sheet seems to be the cause of the reduced thermodynamic stability of M23L-onconase. Work is in progress to characterise other mutant forms of onconase to clarify the molecular determinants of its stability.

[1] Mosimann, S.C., Ardelt, W., James, M.N.G., J.Mol.Biol., 236, 1141-1153, 1994.

[2] Wu, Y., Mikulski, S.M., Ardelt, W., Ryback S.M., Youle, R.J., J.Biol.Chem., 268, 10686-10693, 1993.