Room Temperature Phosphorescence Study of Phosphate Binding in Escherichia Coli Alkaline Phosphatase

Abstract

The phosphorescence spectrum and decay of TrplO9 in Escherichia coli alkaline phosphatase was measured for the enzyme in 10 mM Tris/HCl, pH 7.4, at 21 "C. Changes in the spectrum and decay from the steady-state in response to non-covalent phosphate binding suggested a phosphate-induced alteration in the local environment surrounding Trpl09 which lies buried below the active site. The seemingly inflexible structure in the region of Trpl09, as judged by its very long phosphorescence lifetime, appeared unaltered when the enzyme was symmetrically bound with phosphate. However, the protein with phosphate bound to only one site displayed a marked increase in flexibility that extended over both subunits. For ratios of phosphatelenzyme (mollniol) between 1.0 and 2.0, the observation of exponential phosphorescence decays with lifetimes that are a function of dilution provided evidence for the rapid exchange between phosphate half-saturated and fully-saturated enzymes consistent with observed enzyme turnover rates. The lifetimes under these conditions result in the calculation of a Kd for the dissociation of phosphate from the doubly occupied enzyme of 1.1 ?0.1 pM. The non-exponential decays at P/E, (phosphate/ dimeric enzyme) ratios less than 1.0 revealed that the exchange of phosphate between phosphate-free and half-saturated enzymes was not occurring on the timescale of the phosphorescence decay times, which implied that the half-saturated molecule cannot be contributing significantly to catalysis under steadystate conditions. The observation that the phosphorescence decay at a P/Ed ratio of 1.0 is exponential with a lifetime characteristic of the half-saturated species indicates that the binding of the first phosphate is significantly greater than the second, or that the binding exhibits negative cooperativity.Keywords: alkaline phosphatase; phosphorescence ; phosphate binding ; conformational flexibility ; negative cooperativity.Escherichia coli alkaline phosphatase (AP) is usually considered as a model for the alkaline phosphatases found in a wide variety of species (Wyckoff et al., 1983;Kim and Wyckoff, 1989). The amino acid sequences of the alkaline phosphatases are highly conserved in evolution and the kinetic properties are similar among the enzymes that are widely distributed from bacteria to mammalian tissues . The enzyme is a nonspecific phosphomonoesterase that catalyzes the hydrolysis of phosphate monoesters to the corresponding alcohol, and in addition in the presence of a phosphate acceptor, a transphosphorylation reaction involving a phosphoseryl intermediate (Schwartz and Lipmann, 1961;Dayan and Wilson, 1964;Bradshaw et al., 1981). The dimeric AP molecule has a molecular mass of 95 kDa with two tightly bound zinc ions, and one magnesium ion in each of the active centers of the two identical subunits. Its kinetic properties have been well characterized and it has been established that phosphate dissociation from the enzyme is the rate-limiting step in catalysis at alkaline pH (Reid

Multiple Unfolding Intermediates of Human Placental Alkaline Phosphatase in Equilibrium Urea Denaturation

“…Tryptophanyl fluorescence or phosphorescence has been demonstrated to be excellent structural probe for E. coli alkaline phosphatase (Cioni et al, 1989;Mersol et al, 1991Mersol et al, , 1993Strambini, 1987;Subramaniam et al, 1995;Sun et al, 1997). The four Trp in the placental enzyme are 100% conserved in mammals but not in bacteria.…”

Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli

“…A model describing the catalytic mechanism of APase from E. coli, based on the results of the kinetic experiments and in accordance with the data available in the literature, has been proposed. The model encompasses the experimental data indicating dimer asymmetry [19,26], unequal affinity of subunits for Mg 2+ and P i [6,9,20,24,25,28–31], conformational changes in the catalytic cycle [8,30,32–34], and the role of Mg 2+ in an allosteric activation. Asymmetry is an intrinsic characteristic of dimeric APase, and it is not the consequence of unequal saturation with Mg 2+ .…”

Chloride promotes refolding of active Vibrio alkaline phosphatase through an inactive dimeric intermediate with an altered interface

“…A plausible explanation for the difficulty is that the half‐of‐sites structural oscillation must involve a subtle conformational change that is difficult to detect in a mixed state. During purification, APs usually have a phosphate occupied bound in both active sites, which needs to be removed by denaturation and dialysis before studies on occupancy in relation to crosstalk between subunits can be performed . This is not always possible since demetallization occurring during the process of inactivation and other considerations often make the process irreversible.…”