THE GLY74-]SER AND SER3-]-ALA MUTATIONS IN RHODOBACTER-SPHAEROIDES-Y THIOREDOXIN - EFFECTS ON ACTIVE-SITE REACTIVITY AND PROTEIN-INTERACTION

In this study? we report the effects of two different substitutions in Rhodobacter sphaeroides thioredoxin on two regions of the protein: th e N-terminus end and the hydrophobic area implicated in protein/protei n interactions. We have produced by site-directed mutagenesis R. sphae roides thioredoxin single and double mutants in which the glycine resi due at position 74 is changed to a serine and the serine at position 3 is changed to an alanine; the three mutant proteins have been purifie d. The two substitutions are not equivalent. Substitution of serine by alanine increased the pi from 5.2 to 6.1, this pi value was the same in the double-mutated protein, which demonstrates the presence of a lo cal conformational change. In vivo studies showed that the Gly74-->Ser substitution completely prevented phage T3/7 growth whereas the Ser3- ->Ala substitution had no effect. This finding was corroborated by the large decrease (100-fold) of polymerase activity for the double mutan t in the in vitro measurement of phage T7 DNA polymerase activity with the corresponding pure proteins. Although marginal (within a factor o f two), the effects of the two substitutions on the catalytic activiti es of the thioredoxin reductase reaction confirmed their difference. S ubstitution of serine by alanine had no effect on the K-m and resulted in an improvement in the catalytic efficiency. In contrast, the secon d substitution increased the K-m value, without improving the catalyti c efficiency. The following can be concluded (a) glycine74 of R. sphae roides thioredoxin has a direct role in the binding of T7 gene 5 prote in and the hydrophobic area of thioredoxin; (b) the N-terminus plays a role in maintaining the conformational integrity of the active site; (c) the flexibility of Gly74 in the hydrophobic region involved in pro tein/protein interaction is the operative factor in the case of the ac tivity of thioredoxin in the T7 DNA polymerase.