Probing the Mechanism of the Alkaline Phosphatase Reaction by 67Zn and 25Mg NMR
EMSL Project ID
2585b
Abstract
Alkaline phosphatase (AP) is a ubiquitous enzyme, which is extremely important in mammals. The enzyme catalyzes the hydrolysis of phosphomonoesters providing the cell with the free alcohol and inorganic phosphate. The fatal human hereditary disease hypophosphatasia, caused by a single amino acid substitution, results from the lack of bone AP activity. Based on the availability of reasonable quantities of E. coli AP in highly pure form, and the similarities between the E. coli AP and eukaryotic APs, the E. coli enzyme has been widely used as a model for all APs. The AP from E. coli is a dimer of two identical chains contains 449 amino acids and each active site has binding sites for two Zn2+ and one Mg2+. AP has become a model system for the study of the mechanism of phosphate ester hydrolysis by metalloenzymes. The only structures of AP that have been determined are for the dimeric E. coli and human placenta enzymes. The overall structures of the two enzymes are similar as well as their respective catalytic mechanisms. In fact, the serine that is phosphorylated and the ligands to all three metal sites are conserved. The two zinc binding sites are organized in an almost identical fashion to other metalloenzymes that cleave phosphoesters. For many years the role of Mg2+ in the mechanism was unknown; however, work in our laboratory has firmly established the role of this metal and the requirement for three metals in the active site of APs. In order to fully understand the rate acceleration in the AP reaction it is critical to understand the role that the metals in the active site. The specific aim of this proposal is to investigate the role of the metals in AP for catalysis and structural stabilization by using solid-state 67Zn and 25Mg NMR to directly probe the metal ligation and environment. Here we use NMR techniques to probe how the metals activate the coordinated water molecules for nucleophilic attack. In parallel experiments, not part of this proposal, we will use X-ray crystallography to probe the structural stabilization induced by metal binding and ligation.It has not been possible to directly observe zinc by NMR due to quadrupole relaxation. We will now make use of the new solid-state NMR technique developed by P. Ellis to observe 67Zn directly in the active site using a combination of low-temperature NMR, cross-polarization, spin-echo techniques and utilizing the new low-temperature NMR probe. AP will also become a new model system with which to extend the technique to proteins of larger size (47 KDa/subunit) and to the study of Mg as well as Zn.If the two metal sites cannot be assigned directly based upon differences in quadrupole coupling by comparison to model compounds, we will perturb one of the metal sites either by binding an inhibitor such as mercaptomethylphosponate (MMP), which only interactions with the Zn1 site or a mutant AP that has an alteration in the metal coordination at Zn1 (H412N). 67Zn NMR will be used to detect directly the water coordinated to Zn2 that acts as the nucleophile for the hydrolysis of the phosphoserine intermediate in the second step of the mechanism, using techniques developed for carbonic anhydrase. Experiments will first be conducted at pH values at which the coordinated water should be completely deprotonated (pH 10) or completely protonated (pH 4). Once the quadrupole coupling of the deprotonated and protonated species are assigned, the pH can be varied to determine the pKa of the coordinated water directly. If the Pi-free enzyme is not a good model for this step in the reaction we will use the H331Q mutant AP which has a stable phosphoserine. Similar experiments with 25Mg are planned to provide experimental support for the role of the Mg-coordinated water in the mechanism. Finally, the possible mixed occupancy of the M3 site with zinc and magnesium can be determined directly by the 67Zn NMR experiments.
Project Details
Project type
Capability Research
Start Date
2004-04-16
End Date
2006-04-24
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members