title: Over-the-barrier transition state analogues and crystal structure with Mycobacterium tuberculosis purine nucleoside phosphorylase.
abstract: Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 A) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Lewandowicz
authors.forename: Wuxian
authors.initials: W
authors.lastname: Shi
authors.forename: Gary B
authors.initials: GB
authors.lastname: Evans
authors.forename: Peter C
authors.initials: PC
authors.lastname: Tyler
authors.forename: Richard H
authors.initials: RH
authors.lastname: Furneaux
authors.forename: Luiz A
authors.initials: LA
authors.lastname: Basso
authors.forename: Diogenes S
authors.initials: DS
authors.lastname: Santos
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D007288:Inosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D018716:Molecular Mimicry; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D011684:Purine Nucleosides; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011744:Pyrimidinones / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011758:Pyrroles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D011684:Purine Nucleosides; D011744:Pyrimidinones; D011758:Pyrroles; C113100:immucillin G; C113101:forodesine; D007288:Inosine; D011683:Purine-Nucleoside Phosphorylase