Ping-pong mechanism transferase

The following experiments shed some light. Kinetic studies of sucdnyl-CoA-acetoacetate CoA transferase indicate a ping-pong mechanism. The enzyme alternates between two distinct forms, one of which has been shown to contain bound CoA.932-934 The E-CoA intermediate formed from enzyme plus acetoacetyl-CoA was reduced with 3H-containing sodium borohydride and the protein was completely hydrolyzed with HC1. Tritium-containing a-amino-8-hydroxyvaleric acid was isolated. Since thioesters (as well as oxygen esters) are cleaved in a two-step process... 

In conclusion, all enzymatic methyl transfer reactions studied so far proceed with net inversion of configuration of the methyl group. All these methyl transferases therefore involve an uneven number of transfers of the methyl group, most likely a single, direct transfer from the sulfur of AdoMet to the acceptor atom in an Sj.2-type reaction. Ping-pong mechanisms in which a group in the enzyme active site is transiently methylated can be excluded. The two substrates must be oriented in the enzyme active site such that in the transition state the sulfur, the methyl carbon and the acceptor atom form a linear array. 

Fig. 4. Ether phospholipid synthesis from dihydroxyacetone-phosphate. (A) Dihydroxyacetone-P acyl transferase (DHAPAT). The first step of ether phospholipid synthesis is catalyzed by peroxisomal DHAPAT. This enzyme is a required component of complex ether lipid biosynthesis and its role cannot be assumed by a cytosolic enzyme that also forms acyldihydroxyacetone-P. (B) Ether bond formation by alkyl-DHAP synthase. The reaction that forms the 0-alkyl bond is catalyzed by alkyl-DHAP synthase and is thought to proceed via a ping-pong mechanism. Upon binding of acyl-DHAP to the enzyme alkyl-DHAP synthase, the pro-f hydrogen at carbon atom 1 is exchanged by enolization of the ketone, followed by release of the acyl moiety to form an activated enzyme-DHAP complex. The carbon atom at the 1-position of DHAP in the enzyme complex is thought to carry a positive charge that may be stabilized by an essential sulfhydryl group of the enzyme thus, the incoming alkox-ide ion reacts with carbon atom 1 to form the ether bond of alkyl-DHAP. It has been proposed that a nucleophilic cofactor at the active site covalently binds the DHAP portion of the substrate.

Enzymes with ping-pong mechanisms include various transferases, oxido-reductases, and proteases. The intermediate E in the action of the protease chymotrypsin (Atlas P3), for instance, is formed by modification of a serine residue in the active site. 

Scheme 8 Two mechanistic proposals for the catalytic mechanism of CoA-transferases. In mechanism A, an acyl-enzyme Intermediate Is formed by reaction of an enzyme-bound glutamate (aspartate for Class III enzymes) with the donor acyl-CoA, followed by the formation of an enzyme-bound glutamyl- (or aspartyl-) CoA thioester Intermediate. The thioester subsequently reacts with the acceptor carboxylate to give a new acyl-enzyme anhydride from which the acyl group Is transferred to CoA. In Class I transferases, this process follows classical ping-pong kinetics, whereas In Class III enzymes the donor carboxylate only leaves the enzyme complex upon formation of the product (see text for details). Mechanism B represents a ternary complex mechanism as used by Class II enzymes In which a transient anhydride made up of the donor and acceptor acyl groups Is formed by reaction of the acceptor carboxylate with the donor acyl-ACP. The free ACP subsequently reacts with this anhydride to complete acyl transfer.

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