Mechanistic Studies of the Protonation–Deprotonation Reactions for Type 1 and Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

• Published in: Journal of the American Chemical Society Vol. 140; no. 40; pp. 12900 - 12908
• Main Authors: Neti, Syam Sundar, Pan, Jian-Jung, Poulter, C. Dale
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.10.2018
• Subjects: Deuterium - metabolism; Escherichia coli - enzymology; Escherichia coli - metabolism; Hemiterpenes - metabolism; Isomerases - metabolism; Isomerism; Models, Molecular; Organophosphorus Compounds - metabolism; Protons; Streptococcus pneumoniae - enzymology; Streptococcus pneumoniae - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.8b07274

Type 1 and type 2 isopentenyl diphosphate:​dimethyl­allyl diphosphate isomerase (IDI-1 and IDI-2) catalyze the inter­conversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the fundamental building blocks for biosynthesis of isoprenoid compounds. Previous studies indicate that both isoforms of IDI catalyze isomerization by a protonation–deprotonation mechanism. IDI-1 and IDI-2 are “sluggish” enzymes with turnover times of ∼10 s–1 and ∼1 s–1, respectively. We measured incorporation of deuterium into IPP and DMAPP in D2O buffer for IDI-1 and IDI-2 under conditions where newly synthesized DMAPP is immediately and irreversibly removed by coupling its release to condensation with l-tryptophan catalyzed by dimethyl­allyl­trytophan synthase. During the course of the reactions, we detected formation of d 1, d 2, and d 3 isotopologues of IPP and DMAPP, which were formed during up to five isomerizations between IPP and DMAPP during each turnover. The patterns for deuterium incorporation into IPP show that d 2-IPP is formed in preference to d 1-IPP for both enzymes. Analysis of the patterns of deuterium incorporation are consistent with a mechanism involving addition and removal of protons by a concerted asynchronous process, where addition substantially precedes removal, or a stepwise process through a short-lived (<3 ps) tertiary carbo­cationic intermediate. Previous work with mechanism-based inhibitors and related model studies supports a concerted asynchronous mechanism for the enzyme-catalyzed isomerizations.