Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase

Greg A. Kicska, Peter C. Tyler, Gary B. Evans, Richard H. Furneaux, Wuxian Shi, Alexander Fedorov, Andrzej Lewandowicz, Sean M. Cahill, Steven C. Almo, Vern L. Schramm

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64 Scopus citations


Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (KI* = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP - ImmG - PO4 is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/ acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4′-imino group to a 4′-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pKa changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH3-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by > 107. Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1′-CH2)-ImmH) increased the distance between leaving and oxacarbenium groups and increased Ki to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.

Original languageEnglish (US)
Pages (from-to)14489-14498
Number of pages10
Issue number49
StatePublished - Dec 10 2002

ASJC Scopus subject areas

  • Biochemistry


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