Protein Mass-Modulated Effects in Alkaline Phosphatase

Abstract

Recent experimental studies engaging isotopically substituted protein (heavy protein) have revealed that many, but not all, enzymatic systems exhibit altered chemical steps in response to an altered mass. The results have been interpreted as femtosecond protein dynamics at the active site being linked (or not) to transition-state barrier crossing. An altered enzyme mass can influence several kinetic parameters (k_cat, K_m, and k_chem) in amounts of ≤30% relative to light enzymes. An early report on deuterium-labeled Escherichia coli alkaline phosphatase (AP) showed an unusually large enzyme kinetic isotope effect on k_cat. We examined steady-state and chemical step properties of native AP, [²H]AP, and [²H,¹³C,¹⁵N]AP to characterize the role of heavy enzyme protein dynamics in reactions catalyzed by AP. Both [²H]- and [²H,¹³C,¹⁵N]APs showed unaltered steady-state and single-turnover rate constants. These findings characterize AP as one of the enzymes in which mass-dependent catalytic site dynamics is dominated by reactant-linked atomic motions. Two catalytic site zinc ions activate the oxygen nucleophiles in the catalytic site of AP. The mass of the zinc ions is unchanged in light and heavy APs. They are essentially linked to catalysis and provide a possible explanation for the loss of linkage between catalysis and protein mass in these enzymes.

Figure 1.

(A) Catalytic site configuration for EcAP based on the X-ray crystal structure of the enzyme with inorganic phosphate bound in the active site (Protein Data Bank entry 3TG0). The catalytic site serine is colored green, and the phosphate is colored orange and red. (B) Proposed transition-state structure for EcAP based upon a crystal structure of EcAP covalently bound to a pentavalent vanadate ester. The two Zn²⁺ ions are ~4 Å apart.

Figure 2.

Steady-state substrate saturation curves for (A) pNPP and (B) 4-MUP substrates catalyzed by native EcAP (black), [²H]AP (red), and [²H,¹³C,¹⁵N]AP (blue). Lines are data fit to eq 1.

Figure 3.

Representative stopped-flow traces for single-turnover experiments of 4-methylumbelliferyl phosphate. The fluorescence change resulting from reaction with APs is observed after rapid mixing. The traces were fitted to a single-exponential equation followed by a linear phase to determine the rate of phosphorylation of native AP (black), [²H]AP (red), and [²H,¹³C,¹⁵N]AP (blue). Traces are offset for the purpose of presentation.

Scheme 1.

Simplified Catalytic Mechanism of Alkaline Phosphatase

Scheme 2.

Schematic Representation of the AP-Catalyzed Phosphorylation of 4-Methylumbelliferyl Phosphate^{a a}E, ROP, ROH, E-P, and E·P_i represent the enzymes, substrate 4-methylumbelliferyl phosphate, product 4-methylumbelliferone, covalently phosphorylated enzyme, and noncovalent enzyme phosphate complex, respectively.