ASPARTIC-ACID 26 IN REDUCED ESCHERICHIA-COLI THIOREDOXIN HAS A PK(A)GREATER-THAN-9

Apparent pK(a) values of active site residues Asp26, Cys32, and Cys35 in reduced thioredoxin have been characterized. Both wild-type thiored oxin and mutant D26A thioredoxin were selectively C-13-enriched on cys teine beta-carbons. In both proteins, the variation with pH of (1)HB1, (1)HB2, and (CB)-C-13 NMR chemical shifts has been measured. In wild- type reduced thioredoxin, for both cysteines, the pH versus chemical s hift plots of HB1 protons can be fit to one titration with pK(a) value s of 7.0-7.1. In contrast, the HB2 protons and beta-carbons give pH-ch emical shift plots that clearly reflect more than one titration; fits to the data give apparent pK(a) values of 7.0-7.3 and 9.5 for HB2 prot ons and 7.5-7.9 and 9.2-10.2 for CB carbons. In reduced D26A, all thre e probe chemical shifts have a pH dependence that is fit by one titrat ion with pK(a) of 7.4-7.9. The absence of a titration with pK(a) >9 in D26A, taken together with cysteine thiol pK(a) values of 7.1 and 7.9 determined by Raman spectroscopy [Li er al. (1993) Biochemistry 32, 58 00-5808], indicates that the pK(a) >9 in reduced thioredoxin is that o f Asp26. This is highly significant in view of the previous observatio n that, in oxidized thioredoxin, Asp26 pK(a) is 7.5 [Langsetmo et al. (1991) Biochemistry 30, 7603-7609]. The very high pK(a) values of thes e carboxyls is consistent with their local environment in the three-di mensional structure; the Asp26 side chain in oxidized thioredoxin is a lmost but not completely buried, and in reduced thioredoxin it may be even more buried. The upward shift in pK(a) of Asp26 in reduced thiore doxin accounts for the lower stability of reduced compared to oxidized thioredoxin and suggests that the function of this very highly conser ved active site group is to regulate the redox potential of the enzyme through thermodynamic linkage to global stability. The higher pK(a) o f Asp26 in reduced thioredoxin also offers an explanation for the pref erence for reduced thioredoxin in phage maturation processes.