Trypsin-modified alkaline phosphatase. Formation of apoenzyme monomer and hybrid dimer.

Mg2+ Binding to Alkaline Phosphatase Correlates with Slow Changes in Protein Lability

“…However, it appears more likely that Mg 2+ binding causes long-range intersubunit interactions that change the overall AP structure significantly. This agrees with previous results from experiments with trypsin-modified AP hybrid dimers which revealed that the modified subunit altered the structural and kinetic properties of the other subunit through subunit interactions ( − ). NMR studies of metal hybrid AP dimers, with Zn 2+ bound to one subunit and Cd 2+ bound to the other, have found that Zn 2+ binding caused substantial changes in the chemical shift of the Cd 2+ ligands over 30 Å away at the other active site, again demonstrating long distance subunit interactions ().…”

“…The B sites bind Zn 2+ more strongly in the presence of phosphate and can also bind Mg 2+ at high concentrations (14); however, Mg 2+ normally occupies the C sites. There appears to be significant cooperativity among metal binding sites and between the two subunits (15,(17)(18)(19)(20)(21).…”

“…The B sites bind Zn 2+ more strongly in the presence of phosphate and can also bind Mg 2+ at high concentrations (); however, Mg 2+ normally occupies the C sites. There appears to be significant cooperativity among metal binding sites and between the two subunits ( 15 , − .

1 Ribbon diagram of the AP structure with two Zn 2+ and one Mg 2+ binding sites indicated at the monomer active site ().

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A Comprehensive Enzyme Kinetic Exercise for Biochemistry

“…Several assay conditions exist for hydrolysis of p -nitrophenyl phosphate including the buffer, wavelength of observation, and absorptivity. Early on, we chose the conveniently available Tris buffer, the wavelength of 410 nm and absorptivity of 16.2 μmol –1 cm –1 mL . The effect of disodium phosphate as product or inhibitor on buffer pH is less than 0.4%.…”

“…The relatively inexpensive bovine (Sigma P7640) enzyme with ≥10 units/mg at 37 °C in diethanolamine at pH 9.8 was used. This enzyme with extinction coefficient E 278 1% = 7.2 and monomer size of 64 kDa is a metallohydrolase homodimer containing two zinc ions that catalytically chelate the phosphate group and one stabilizing magnesium binding site. The reaction (Scheme ) was monitored at 410 nm for production of the p -nitrophenolate ion from the Sigma substrate P4744; product concentration was determined based on an absorptivity of 16.2 μmol –1 cm –1 mL in a 1.0 M Tris pH 10.0 buffer.…”