Mode of Inhibition of β-Hydroxydecanoyl Thioester Dehydrase by 3-Decynoyl-N-acetylcysteamine

Structure of a dehydratase–isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: two catalytic activities in one active site

“…Asp84′, which was not anticipated to be a catalytic residue, is in direct contact with the C-3/C-4 region of the inactivator. Since His70 was identified as the site of covalent modification by 3-decynoyl NAC [18], studies of the mechanism of dehydrase have focused on this residue. Modification of His70 at N⑀2 was demonstrated by 15 N NMR [7].…”

“…Both reactions catalyzed by dehydrase require deprotonation of a relatively non-acidic substrate without the help of a metal ion cofactor. The deprotonation at C-2 of the substrate has been shown to be the rate-determining step in dehydration [17] and in the mechanism-based transformation of 3-decynoyl-NAC into the reactive allene [18]. Questions remain as to the nature of groups other than His70 that participate in catalysis.…”

Kinetics of suicide substrates

“…The specific inactivation of enzymes often lies at the root of effective drug action, and it is, moreover, an important step in the study of the enzyme's active site. Certain substrates (suicide substrates, mechanism-based inactivators) undergo a structural change during the course of catalysis that results in their being bound to the enzyme and inactivating it (Endo et al, 1970;Abeles & Maycock, 1976;Rando, 1977;Walsh et al, 1978;Seiler et al, 1978). It is characteristic of suicide substrates that formation of product and inactivation of enzyme proceed concurrently.…”

Trapping the Complex Molecular Machinery of Polyketide and Fatty Acid Synthases with Tunable Silylcyanohydrin Crosslinkers

“…[14] Once appended, the reactive unit of the resulting crypto-ACP 12 could be revealed by addition of fluoride to afford the corresponding cyanohydrin crypto-ACP 13 in the presence of aD H. Cyanohydrin 13 can then bind to the DH, forming complex 14.L oss of HCN would generate ketone 15,w hich is set to trap ap roximal His residue (Supporting Information, Figure S1b) and form crosslinked 16. [15] Our plan was to first use the fluorescent probes 6-11 [16] to optimize covalent modification of the DH and then return to 3-5 to implement the crosslinking process (15 and 16, Scheme 1). Adapting established methods for pantethein-Scheme 1.…”

“…After deprotection, 13 binds to the DH and inserts its cargo into the DH active site, as shown in 14.H CN is eliminated,a nd the resulting complex 15 can covalentlym odify proximal His residues (Supporting Information, Figure S1). [15] Alternatively,the silylcyanohydrin in 12 can bind to the DH, and the subsequent steps could occur in the DH active site.…”