“Catalysis by Alkaline Phosphatase is Ultrasensitive to Charge Sequestered Between the Active Site Zinc Ions in the Transition State”
نویسندگان
چکیده
The following Supporting Methods describe determination of the charge density on each nonbridging oxygen atom and catalytic proficiencies for the R166S AP-catalyzed reactions of the following substrates: para-nitrophenyl phosphate (pNPP), para-nitrophenyl phosphorothioate (pNPPS), bis-(para-nitrophenyl) phosphate (bpNPP), methyl para-nitrophenyl phosphate (MpNPP), methyl para-nitrophenyl phosphorothioate (MpNPPS), para-nitrophenyl sulfate (pNPS) and di-ethyl para-nitrophenyl phosphate (dEpNPP). These values are summarized in Table 1 in the main text and used in the correlation of Fig. 1 in the main text.
منابع مشابه
Alkaline phosphatase catalysis is ultrasensitive to charge sequestered between the active site zinc ions.
Escherichia coli alkaline phosphatase (AP) is a prototypical bimetalloenzyme, facilitating catalysis of phosphate monoester hydrolysis with two Zn2+ metal ions that are only 4 A apart. In the reaction's transition state, one of the nonbridging oxygen atoms of the transferred group appears to interact directly with the Zn2+ ion metallocluster. To determine the importance and the energetic proper...
متن کاملKinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis.
Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject o...
متن کاملA model of the transition state in the alkaline phosphatase reaction.
A high resolution crystal structure of Escherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 A resolution. The vanadate ion takes on a trigonal bipyramidal geometry and is covalently bound by the active site serine nucleophile. A coordinated water molecule occupies the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the ...
متن کاملProbing the origin of the compromised catalysis of E. coli alkaline phosphatase in its promiscuous sulfatase reaction.
The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here, we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The p...
متن کاملQuantum Mechanical Approach for the Catalytic Mechanism of Dinuclear Zinc Metallo-β-lactamase by Penicillin and Cephalexin: Kinetic and Thermodynamic Points of View
Metallo-β-lactamases (MβL) catalyzing the hydrolytic cleavage of the four-membered β-lactam ring in broad spectrum of antibiotics and therefore inactivating the drug; However, the mechanism of these enzymes is still not well understood. Electronic structure and electronic energy of metallo-β-lactamase active center, two inhibitors of this enzyme including penicillin and cephalexin, and differen...
متن کامل