Tryptophan-Free Human PNP Reveals Catalytic Site Interactions

Abstract

Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Trp residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The (13)C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steady-state kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate-phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Trp residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.

Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase

“…Single-turnover catalytic site rate constants were determined by monitoring the increase in fluorescence upon formation of enzyme-bound guanine (25) in an SX-20 stopped-flow spectrofluorometer (Applied Photophysics; dead time ≤1.25 ms) at 25°C. The excitation wavelength was 280 nm with slit width of 1 mm, and a fluorescence signal above 305 nm (with slit width of 1 mm) was selected using a WG305 Scott filter positioned between the photomultiplier and the sample cell.…”

Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

“…Single-turnover rate constants were determined by monitoring the increase in fluorescence upon formation of enzyme-bound guanine (44), or the decrease in absorption upon conversion of inosine to hypoxanthine in an SX-20 stopped-flow spectrofluorometer (Applied Photophysics) (dead time ≤1.25 ms) at 25°C. For experiments with guanosine, the excitation wavelength was 280 nm with slit width of 0.5 mm, and fluorescence signal above 305 nm (with slit width of 2 mm) was selected using a WG305 Scott filter positioned between the photomultiplier and the sample cell.…”

Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase

“…PNP was used in a design program to alter the catalytic-site response to heavyatom substitution in the enzyme protein. Native PNP exhibits slowed chemistry when made heavy with 2 H, 13 C, and 15 N. We succeeded in designing a second-sphere mutation with improved promoting vibrations to catalyze faster chemistry in response to heavy PNP.…”

“…1) (13). The purine leaving group and phosphate nucleophile are both activated by the enzyme, distorting the symmetry of the normal modes for phosphate and causing the bound purine to exhibit altered spectral properties (14,15). In the femtosecond time period approaching the TS, His257 is hydrogen bonded to the ribosyl 5′-hydroxyl group and directs O5′ toward the O4′ of the purine ring, thus destabilizing the ribosidic bond to facilitate departure of the purine leaving group toward the TS (16).…”

“…Heavy PNP labeled with 15 N, 13 C, and nonexchangeable 2 H has slowed pre-steady-state catalytic-site chemistry and has a lower probability of barrier crossing, but unchanged steady-state kinetic properties (2). Slowed chemistry in heavy PNP has been interpreted as a mass-dependent slowed femtosecond dynamical search for the catalytic-site geometry permitting TS formation and barrier crossing.…”