iBet uBet web content aggregator. Adding the entire web to your favor.
iBet uBet web content aggregator. Adding the entire web to your favor.



Link to original content: https://pubmed.ncbi.nlm.nih.gov/17630738/
Kinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis - PubMed Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Aug 8;129(31):9789-98.
doi: 10.1021/ja072196+. Epub 2007 Jul 14.

Kinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis

Jesse G Zalatan  1 , Irina Catrina, Rebecca Mitchell, Piotr K Grzyska, Patrick J O'brien, Daniel Herschlag, Alvan C Hengge
Affiliations

Kinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis

Jesse G Zalatan et al. J Am Chem Soc. .

Abstract

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 of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active-site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Phosphate and phosphorothioate esters for which isotope effects are reported: red, nonbridge phosphoryl oxygen atoms [18(V/K)nonbridge]; blue, bridge oxygen atom, site of bond fission [18(V/K)bridge]; green, nitrogen atom of the leaving group [15(V/K)]. The structures are depicted with partial double bonds to emphasize that the nonbridging oxygen atoms that contribute to 18(V/K)nonbridge are formally equivalent when the substrates are free in solution. For simplicity, the P-S and P-O bonds of pNPPS are depicted with the same partial double bond character, although it is likely that the P-S bond has more single bond character than the P-O bonds.,
Figure 2
Figure 2
Compilation of kinetic data for the reactions of wt and R166S AP, represented by closed and open symbols, respectively. Reactions of alkyl phosphates (■, □) give values of βlg of −0.85 ± 0.1 and −0.66 ± 0.1 respectively for reactions with wt and R166S AP., Reactions of aryl phosphates (♦) with wt AP are limited by a non-chemical step (dashed line). The rate constant for reaction of the aryl phosphate pNPP2− (◊) with R166S AP deviates negatively from the value expected by extrapolation from the line for alkyl phosphates (dotted line and single-headed arrow). Rate constants for the reactions of mNBP2− with wt and R166S AP (●, ○) deviate positively from the values expected from the line for alkyl phosphates (double-headed arrows).
Figure 3
Figure 3
KIEs for AP-catalyzed reactions are different from the corresponding solution reaction. A) Predicted direction of changes in KIEs relative to the solution reaction for two different models for behavior of phosphate monoester substrates in the AP active site. In model 1, the transition state is loose, similar to that in solution, and strong interactions with active site metal ions lead to decreases in both 18(V/K)bridge and 18(V/K)nonbridge. In model 2, AP catalyzes phosphate monoester hydrolysis through a tighter transition state than that in solution. This model predicts a decrease in 18(V/K)bridge and an increase in 18(V/K)nonbridge based on comparisons to corresponding solution reactions. B) The observed values of both 18(V/K)bridge and 18(V/K)nonbridge for AP-catalyzed pNPP2− hydrolysis decrease relative to the corresponding solution reaction, consistent with model 2. Isotope effects are expressed as a percentage difference from unity [KIE% = (KIEobs −1)*100]. Values for the nonenzymatic reaction are shown in black and values for the AP-catalyzed reaction are shown in grey.
Scheme 1
Scheme 1
Transition state model for phosphoryl transfer catalyzed by AP based on the structure with a bound vanadate ligand.,
Scheme 2
Scheme 2
Phosphate monoester hydrolysis through a loose transition state.
Scheme 3
Scheme 3
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lad C, Williams NH, Wolfenden R. Proc. Natl. Acad. Sci. U.S.A. 2003;100:5607–5610. - PMC - PubMed
    1. Polanyi MZ. Elektrochem. Z. 1921;27:142.
    1. Pauling L. Chem. Eng. News. 1946;24:1375–1377.
    1. Wolfenden R. Acc. Chem. Res. 1972;5:10–18.
    1. Lienhard GE. Science. 1973;180:149–154. - PubMed

Publication types