Loop dynamics and ligand binding kinetics in the reaction catalyzed by the Yersinia protein tyrosine phosphatase.
The Yersinia protein tyrosine phosphatase (YopH) contains a loop of ten amino acids (the WPD loop) that covers the entrance of the active site of the enzyme during substrate binding. In this work the substrate mimicking competitive inhibitor p-nitrocatechol sulfate (PNC) is used as a probe of the active site. The dynamics of the WPD loop was determined by subjecting an equilibrated system containing YopH, PNC, and YopH bound to PNC to a laser induced temperature jump, and subsequently following the change in equilibrium due to the perturbation. Using this methodology the dynamics associated with substrate binding in YopH have been determined. These results indicate that substrate binding is coupled to the WPD loop motion, and WPD loop dynamics occur in the sub-millisecond time scale. The significance of these dynamic results is interpreted in terms of the catalytic cycle of the enzyme.
Khajehpour
Mazdak
M
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. mazdak@medusa.bioc.aecom.yu.edu
Wu
Li
L
Liu
Sijiu
S
Zhadin
Nick
N
Zhang
Zhong-Yin
ZY
Callender
Robert
R
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D020134:Catalytic Domain; D002396:Catechols / Q000737:chemistry / Q000378:metabolism; D003460:Crystallization; D003545:Cysteine / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D007834:Lasers; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D015007:Yersinia / Q000201:enzymology*
D002396:Catechols; D008024:Ligands; D001224:Aspartic Acid; D014364:Tryptophan; D017027:Protein Tyrosine Phosphatases; C021228:4-nitrocatechol sulfate; D003545:Cysteine