Diversity of chemical mechanisms in thioredoxin catalysis revealed by single-molecule force spectroscopy.
Thioredoxins (Trxs) are oxidoreductase enzymes, present in all organisms, that catalyze the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single-molecule level. Here we use single-molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different Trx enzymes. All Trxs show a characteristic Michaelis-Menten mechanism that is detected when the disulfide bond is stretched at low forces, but at high forces, two different chemical behaviors distinguish bacterial-origin from eukaryotic-origin Trxs. Eukaryotic-origin Trxs reduce disulfide bonds through a single-electron transfer reaction (SET), whereas bacterial-origin Trxs show both nucleophilic substitution (S(N)2) and SET reactions. A computational analysis of Trx structures identifies the evolution of the binding groove as an important factor controlling the chemistry of Trx catalysis.
Perez-Jimenez
Raul
R
Department of Biological Sciences, Columbia University, New York, New York, USA. raulpjc@biology.columbia.edu
Li
Jingyuan
J
Kosuri
Pallav
P
Sanchez-Romero
Inmaculada
I
Wiita
Arun P
AP
Rodriguez-Larrea
David
D
Chueca
Ana
A
Holmgren
Arne
A
Miranda-Vizuete
Antonio
A
Becker
Katja
K
Cho
Seung-Hyun
SH
Beckwith
Jon
J
Gelhaye
Eric
E
Jacquot
Jean P
JP
Gaucher
Eric A
EA
Gaucher
Eric
E
Sanchez-Ruiz
Jose M
JM
Berne
Bruce J
BJ
Fernandez
Julio M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D019143:Evolution, Molecular; D014644:Genetic Variation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000145:classification / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004220:Disulfides; D007527:Isoenzymes; D013879:Thioredoxins; D010088:Oxidoreductases