Nucleotidyl cyclases

Sinha, S. C., and Sprang, S. R. (2006). Structures, mechanism, regulation and evolution of class III nucleotidyl cyclases. Rev. Physiol. Biochem. Pharmacol. 157, 105-140. 

Poeggel G, Luppa H (1988) Histochemistry of nucleotidyl cyclases and cyclic nucleotide phosphodiesterases. Histochem. J., 20. 249-268. 

D Nucleotidyl cyclases Nucleoside triphosphate cydisation under formation of pyrophosphate 4.6.1. Phosphorous oxygen lyases... 

Nucleotidyl cyclases catalyse the formation of 3, 5 cyclic nucleotide monophosphates from nucleotide triphosphates. Adenylate cyclase, which catalyses the formation of cAMP, is of particular importance because of its major biological role in regulating the level of cAMP and hence regulating cellular communication. Study of the mechanism of enzyme-catalysed cycli-zation has been hindered through the lack of pure enzyme preparations— adenylate cyclases are membrane-bound proteins. However, Coderre and Gerlt have completed stereochemical analyses of the accessible adenylate cyclase from Brevibacterium liquefaciens. 

The concentration of cyclic nucleotides in biological systems is regulated by synthesis (by nucleotidyl cyclases) and degradation (by cyclic-nucleotide phosphodiesterases). cAMP phosphodiesterase catalyses the hydrolysis of cAMP to AMP and thus is responsible for terminating the activity of cAMP in vivo. 

Note the multiple receptor sites on nucleotidyl cyclase and the membranous location of the low Km phosphodiesterase. Two bound (one membrane-associated) euid one free pools of the cyclic nucleotides are also shown. 

Few details are available about the chemical mechanisms of the reactions catalyzed by nucleotidyl cyclases since these enzymes can be associated with membranes and therefore difficult to purify and/or are present in very small amounts and therefore difficult to obtain in amounts compatible with many modem enzymological techniques. We have decided to direct our attention to bacterial adenylate cyclases because one, the enzyme produced by Brevi-bacterium liquefaciens, has been purified to homogeneity (Takai et al., 1974) and the gene for another, that from Salmonella typhimurium. has been cloned in a multiple-copy plasmid (Wang et ai, 1981). Given the limited amounts of enzymes that are presently available, chemical studies designed to ascertain directly if the reaction occurs via formation and breakdown of an adenylated enzyme intermediate are impossible therefore, we decided to use a stereochemical approach to obtain information regarding the existence of an adenylated enzyme intermediate in the reaction catalyzed by the cyclase isolated from B. liquefaciens. 

Eckstein s study of the stereochemical course of a bovine guanylate cyclase utilized chemically synthesized [a- 0, 0]GTP as substrate, and the configuration of the product cyclic [ 0, 0]GMP was determined by P NMR following methylation, as described in Section III,A. This nucleotidyl cyclase was also found to proceed with inversion of configuration at phosphorus (Senter et a/., 1983). 

Using two enzymes, a mammalian adenylate cyclase and myosin ATPase, as examples the application of phosphorothioate analogues to the study of the mechanism of nucleotidyl and phosphoryl transfer will be described. 

Adenylyl and guanylyl cyclases catalyze their respective cyclization reactions with inversion of configuration at P of ATP or GTP (55-59). There is no other evidence for the involvement of a nucleotidyl-enzyme in these reactions. Therefore, the mechanism apparently does not involve nucleophilic catalysis by the enzyme, but instead proceeds with intramolecular nucleophilic displacement of MgPPi from P of the substrate by the 3 -hydroxyl group. This forms cAMP or cGMP and MgPPj in an in-line reaction with a single displacement at phosphorus. 

Enzymes that catalyze nucleotidyl transfer reactions and utilize acceptors other than water involve the interconversion of prochiral substrates and products. These enzymes include the nucleotidyl polymerases and cyclases... 

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