Tryptophan-free human PNP reveals catalytic site interactions.
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.
Ghanem
Mahmoud
M
Departments of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Saen-oon
Suwipa
S
Zhadin
Nickolay
N
Wing
Corin
C
Cahill
Sean M
SM
Schwartz
Steven D
SD
Callender
Robert
R
Schramm
Vern L
VL
D001665:Binding Sites; D020134:Catalytic Domain; D006147:Guanine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism*
D010710:Phosphates; D019271:Hypoxanthine; D006147:Guanine; D014364:Tryptophan; D011683:Purine-Nucleoside Phosphorylase