Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases.
Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X-ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions. The structural analysis is supported by thermodynamic and kinetic data. The AP catalysis essentially requires octahedral coordination in the M3 site, but stability is adjusted with the conformational freedom of the metal ion. Comparison with the mesophilic Escherichia coli, AP shows differences in the charge transfer network in providing the chemically optimal metal combination for catalysis. Our results provide explanation why the TAB5 and E. coli APs respond in an opposite way to mutagenesis in their active sites. They provide a lesson on chemical fine tuning and the importance of the second coordination sphere in defining metal specificity in enzymes. Understanding the framework of AP catalysis is essential in the efforts to design even more powerful tools for modern biotechnology.
Koutsioulis
Dimitris
D
New England Biolabs Inc., Ipswich, Massachusetts 01938-2723, USA.
Lyskowski
Andrzej
A
Mäki
Seija
S
Guthrie
Ellen
E
Feller
Georges
G
Bouriotis
Vassilis
V
Heikinheimo
Pirkko
P
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D017510:Protein Folding; D013696:Temperature; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; D008670:Metals; D000469:Alkaline Phosphatase; D015032:Zinc