Preparation and Characterization of Oxoiron(IV) Chlorin Complexes as the First Models for a Reaction Intermediate in the Catalytic Cycle of Cytochrome d

1994 ◽  
Vol 116 (2) ◽  
pp. 634-641 ◽  
Author(s):  
Shinji Ozawa ◽  
Yoshihito Watanabe ◽  
Satoru Nakashima ◽  
Teizo Kitagawa ◽  
Isao Morishima
Keyword(s):  
Catalytic Cycle ◽  
Reaction Intermediate ◽  
Cytochrome D

Isolation and characterization of a peroxo manganese (III) dioxygen reaction intermediate using cryogenic ion vibrational predissociation spectroscopy

2013 ◽  
Vol 354-355 ◽  
pp. 33-38 ◽  
Author(s):  
Arron B. Wolk ◽  
Christopher M. Leavitt ◽  
Joseph A. Fournier ◽  
Michael Z. Kamrath ◽  
Gayan B. Wijeratne ◽  
...  
Keyword(s):  
Vibrational Predissociation ◽  
Reaction Intermediate ◽  
Isolation And Characterization

scholarly journals Characterization of the Catalytic Cycle of ATP Hydrolysis by Human P-glycoprotein

2001 ◽  
Vol 276 (15) ◽  
pp. 11653-11661 ◽  
Author(s):  
Zuben E. Sauna ◽  
Suresh V. Ambudkar
Keyword(s):  
Multidrug Resistance ◽  
Functional Outcomes ◽  
Atp Hydrolysis ◽  
Substrate Binding ◽  
Nucleotide Binding ◽  
Catalytic Cycle ◽  
Nucleotide Binding Sites ◽  
P Glycoprotein ◽  
Random Manner

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000)Proc. Natl. Acad. Sci. U. S. A.97, 2515–2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomesvis à visthe substrate, they show comparablet12for both incorporation and release of nucleotide, and the affinities for [α-32P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [α-32P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.

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