RNase modification, conformational

The single trytophan residue in the native RNase Tt seems to be embedded in the interior of the molecule and cannot be modified by specific reagents. The modification of the residue in 8M urea was carried out with 2-hydroxyl-5-nitrobenzyl bromide by Takahashi (64a) and with A-bromosuccinimide by Kawashima and Ando (65). In both cases complete inactivation occurred when the tryptophan residue was completely modified. However, RNase T, modified with 2-hydroxyl-5-nitrobenzyl bromide in 4 M urea retained about 30% of the original activity (66). Thus, it may be concluded that the tryptophan residue does not participate directly in the catalytic process, since it is situated quite close to the essential Glu 58, it may indirectly participate in the building up of the active conformation (11). 

C/modification) to both DNA and RNA (356). This modification has also been claimed to activate RNase H (367) as a mixed oligomer with DNA and not as a chimera involving de-oxy gaps. This claim is questionable on the basis of conformational and steric factors associated with this modification. 

Amino acid esterases were generated from RNase by using a variety of indole derivatives as modifiers and, perturbing the conformation by titration to an acid pH.23 29 Crosslinking with glutaraldehyde was used to stabilize this new conformation. When assayed with L-tryptophan ethyl ester, the modified RNase was found to possess two pH optima one at 6 and the other at 7.5. Purification of the crude reaction mixture demonstrated the presence of two types of amino acid esterases which account for the two pH optima. After the conformational modification process, the native activity of RNase is lower. After purification of the crude mixture no native RNase activity can be measured in the fractions containing amino acid esterase activity. 

CCMPs have been prepared, which have the ability to catalyze the hydrolysis of organofluorophosphates such as diisopropylphosphorofluoridate and phenylmethylsulfonylfluoride, using several different starting proteins. In addition, semisynthetic fluorohydrolases prepared by the conformational modification of RNase with HMPA were crosslinked with diimidates of chain lengths from Ci to Ce to determine the optimum crosslink for the maximum fluorohydrolase activity. 

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