Second-sphere amino acids contribute to transition-state structure in bovine purine nucleoside phosphorylase.
Transition-state structures of human and bovine of purine nucleoside phosphorylases differ, despite 87% homologous amino acid sequences. Human PNP (HsPNP) has a fully dissociated transition state, while that for bovine PNP (BtPNP) has early SN1 character. Crystal structures and sequence alignment indicate that the active sites of these enzymes are the same within crystallographic analysis, but residues in the second-sphere from the active sites differ significantly. Residues in BtPNP have been mutated toward HsPNP, resulting in double (Asn123Lys; Arg210Gln) and triple mutant PNPs (Val39Thr; Asn123Lys; Arg210Gln). Steady-state kinetic studies indicated unchanged catalytic activity, while pre-steady-state studies indicate that the chemical step is slower in the triple mutant. The mutant enzymes have higher affinity for inhibitors that are mimics of a late dissociative transition state. Kinetic isotope effects (KIEs) and computational chemistry were used to identify the transition-state structure of the triple mutant. Intrinsic KIEs from [1'-3H], [1'-14C], [2'-3H], [5'-3H], and [9-15N] inosines were 1.221, 1.035, 1.073, 1.062 and 1.025, respectively. The primary intrinsic [1'-14C] and [9-15N] KIEs indicate a highly dissociative SN1 transition state with low bond order to the leaving group, a transition state different from the native enzyme. The [1'-14C] KIE suggests significant nucleophilic participation at the transition state. The transition-state structure of triple mutant PNP is altered as a consequence of the amino acids in the second sphere from the catalytic site. These residues are implicated in linking the dynamic motion of the protein to formation of the transition state.
Li
Lei
L
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Luo
Minkui
M
Ghanem
Mahmoud
M
Taylor
Erika A
EA
Schramm
Vern L
VL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D044366:Transition Temperature
D000596:Amino Acids; D011994:Recombinant Proteins; D011683:Purine-Nucleoside Phosphorylase