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The concerted action of a positive charge and hydrogen bonds dynamically regulates the p K a of the nucleophilic cysteine in the NrdH‐redoxin family
Author(s) -
Van Laer Koen,
Oliveira Margarida,
Wahni Khadija,
Messens Joris
Publication year - 2014
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2397
Subject(s) - nucleophile , cysteine , charge (physics) , hydrogen bond , chemistry , action (physics) , stereochemistry , physics , biochemistry , enzyme , catalysis , organic chemistry , molecule , quantum mechanics
NrdH‐redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH‐redoxins have a CVQC active site motif and belong to the thioredoxin‐fold protein family. As for other thioredoxin‐fold proteins, the p K a of the nucleophilic cysteine of NrdH‐redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the p K a value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH‐redoxins were determined, but structural insights explaining the relatively low p K a remained elusive. We subjected C. glutamicum NrdH‐redoxin to an extensive molecular dynamics simulation to expose the factors regulating the p K a of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N‐terminal of the active site further lowers the cysteine p K a . However, site‐directed mutagenesis data show that the major contribution to the lowering of the cysteine p K a comes from the positive charge of the lysine and not from the additional Lys‐Cys hydrogen bond. In 12% of the NrdH‐redoxin family, this lysine is replaced by an arginine that also lowers the cysteine p K a . All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N‐terminally of the active site dynamically regulate the p K a of the nucleophilic cysteine in NrdH‐redoxins.