Aspartic acid 26 in reduced Escherichia coli thioredoxin has a pKa > 9.
Apparent pKa values of active site residues Asp26, Cys32, and Cys35 in reduced thioredoxin have been characterized. Both wild-type thioredoxin and mutant D26A thioredoxin were selectively 13C-enriched on cysteine beta-carbons. In both proteins, the variation with pH of 1HB1, 1HB2, and 13CB NMR chemical shifts has been measured. In wild-type reduced thioredoxin, for both cysteines, the pH versus chemical shift plots of HB1 protons can be fit to one titration with pKa values of 7.0-7.1. In contrast, the HB2 protons and beta-carbons give pH--chemical shift plots that clearly reflect more than one titration; fits to the data give apparent pKa values of 7.0-7.3 and 9.5 for HB2 protons and 7.5-7.9 and 9.2-10.2 for CB carbons. In reduced D26A, all three probe chemical shifts have a pH dependence that is fit by one titration with pKa of 7.4-7.9. The absence of a titration with pKa > 9 in D26A, taken together with cysteine thiol pKa values of 7.1 and 7.9 determined by Raman spectroscopy [Li et al. (1993) Biochemistry 32, 5800-5808], indicates that the pKa > 9 in reduced thioredoxin is that of Asp26. This is highly significant in view of the previous observation that, in oxidized thioredoxin, Asp26 pKa is 7.5 [Langsetmo et al. (1991) Biochemistry 30, 7603-7609]. The very high pKa values of these carboxyls is consistent with their local environment in the three-dimensional structure; the Asp26 side chain in oxidized thioredoxin is almost but not completely buried, and in reduced thioredoxin it may be even more buried.(ABSTRACT TRUNCATED AT 250 WORDS)
Wilson
N A
NA
Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
Barbar
E
E
Fuchs
J A
JA
Woodward
C
C
D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004355:Drug Stability; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D017354:Point Mutation; D011487:Protein Conformation; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics
D001224:Aspartic Acid; D013879:Thioredoxins; D003545:Cysteine