Second-sphere amino acids contribute to transition-state structure in bovine purine nucleoside phosphorylase

Lei Li, Minkui Luo, Mahmoud Ghanem, Erika A. Taylor, Vern L. Schramm

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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 presteady-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.

Original languageEnglish (US)
Pages (from-to)2577-2583
Number of pages7
JournalBiochemistry
Volume47
Issue number8
DOIs
StatePublished - Feb 26 2008

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Purine-Nucleoside Phosphorylase
Isotopes
Amino Acids
Kinetics
Catalytic Domain
Enzymes
Computational chemistry
Amino Acid Sequence Homology
Inosine
Sequence Alignment
Catalyst activity
Crystal structure
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Second-sphere amino acids contribute to transition-state structure in bovine purine nucleoside phosphorylase. / Li, Lei; Luo, Minkui; Ghanem, Mahmoud; Taylor, Erika A.; Schramm, Vern L.

In: Biochemistry, Vol. 47, No. 8, 26.02.2008, p. 2577-2583.

Research output: Contribution to journalArticle

Li, Lei ; Luo, Minkui ; Ghanem, Mahmoud ; Taylor, Erika A. ; Schramm, Vern L. / Second-sphere amino acids contribute to transition-state structure in bovine purine nucleoside phosphorylase. In: Biochemistry. 2008 ; Vol. 47, No. 8. pp. 2577-2583.
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abstract = "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 presteady-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.",
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AB - 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 presteady-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.

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