Coenzyme A Hemithioacetals as Easily Prepared Inhibitors of CoA Ester-Utilizing Enzymes

Abstract

Hemithioacetals are formed by reactions of coenzyme A (CoA) with aldehydes in aqueous solution. Equilibria for hemithioacetal formation with four commercially available aldehydes and rate constants for hemithioacetal dissociation have been studied. The hemithioacetals are viewed as acyl-CoA analogs having a tetrahedral center in place of the planar trigonal thioester carbonyl carbon. These compounds may serve as mimics of the tetrahedral intermediate or transition state in the reactions of acyl-CoA dependent acyltransferase enzymes. The hemithioacetal generated by reaction of CoA with formaldehyde is a poor inhibitor of chloramphenicol acetyltransferase, with a K i more than 6-fold higher than the K m for the substrate acetyl-CoA. The hemithioacetals formed by reaction of CoA with acetaldehyde and trifluroacetaldehyde are substantially better inhibitors, with K i values approximately 2.4-fold and 10-fold lower than the K m values for acetyl-CoA, respectively. The hemithioacetal formed by reaction of CoA with succinic semialdehyde inhibits succinic thiokinase, with a K i 4-fold lower than the K m for the substrate succinyl-CoA. The CoA hemithioacetals provide a novel readily accessible new class of acyl-CoA analogs for use in mechanistic and structural studies of CoA ester-utilizing enzymes.

Mechanistic Investigations of Ruthenium Catalyzed Dehydrogenative Thioester Synthesis and Thioester Hydrogenation

“…Thus, it is unlikely that the reaction proceeds through the coupling of aldehyde and thiol to form a transient hemithioacetal ( Figure 6 ). 40 , 41 In addition to the likely low concentration of aldehyde present in the catalytic system, we note that the formation of a hemithioacetal from aldehyde and thiol is thermodynamically uphill (Δ G = +5.4 kcal/mol), and we also find no kinetically reasonable pathways to dehydrogenate the hemithioacetal to afford the product thioester (most notably, dehydrogenation at Ru-3 via TS 3,2 , Figure 6 ). This conclusion contrasts our original proposal, but is in accord with other related dehydrogenative coupling mechanisms.…”

Molecular Modeling of the Reaction Pathway and Hydride Transfer Reactions of HMG-CoA Reductase

“…Consequently, the opening/closing process of the flap domain and cofactor exchange must be faster than hemithioacetal breakdown after the thioester is reduced. By destabilizing the aldehyde, the enzyme is able to slow down the hemithioacetal breakdown reaction, which is rapid in solution with CoA thiols, 75 to a point where the cofactor exchange is faster. The redox state of the cofactor could also play a role in limiting hemithioacetal breakdown.…”

Coenzyme A Analogues and Derivatives: Synthesis and Applications as Mechanistic Probes of Coenzyme A Ester-Utilizing Enzymes

“…The most easily derived analogues of CoA esters are the hemithioacetals 111 . Four hemithioacetals of CoA were generated by mixing CoA with the appropriate aldehyde 110 (Figure ) . The rates and equilibria of hemithioacetal formation and dissociation were studied.…”

“…Formation and dissociation of the hemithioacetals with acetaldehyde ( 111b ) and succinic semialdehyde ( 111d ) were too fast to measure by simple methods, while the formaldehyde ( 111a ) and trifluoroacetaldehyde ( 111c ) hemithioacetals had rate constants for dissociation of 1.3 × 10 -2 and 3.0 × 10 -3 s -1 , respectively. 111a − c were tested as inhibitors of chloramphenicol acetyltransferase . Conditions were chosen such that inhibition by CoA and aldehyde was small relative to inhibition by the hemithioacetal.…”

“…Four hemithioacetals of CoA were generated by mixing CoA with the appropriate aldehyde 110 (Figure 34). 231 The rates and equilibria of hemithioacetal formation and dissociation were studied. Formation and dissociation of the hemithioacetals with acetaldehyde (111b) and succinic semialdehyde (111d) were too fast to measure by simple methods, while the formaldehyde (111a) and trifluoroacetaldehyde (111c) hemithioacetals had rate constants for dissociation of 1.3 × 10 -2 and 3.0 × 10 -3 s -1 , respectively.…”