Unifying mechanism for Aplysia ADP-ribosyl cyclase and CD38/NAD(+) glycohydrolases

Highly purified Aplysia californica ADP-ribosyl cyclase was found to be a m ultifunctional enzyme. In addition to the known transformation of NAD(+) in to cADP-ribose this enzyme is able to catalyst: the solvolysis (hydrolysis and methanolysis) of cADP-ribose. This cADP-ribose hydrolase activity, whic h becomes detectable only at high concentrations of the enzyme, is amplifie d with analogues such as pyridine adenine dinucleotide, in which the cleava ge rate of the pyridinium-ribose bond is much reduced compared with NAD(+). Although the specificity ratio V-max/K-m is in favour of NAD(+) by 4 order s of magnitude, this multifunctionality allowed us to propose a 'partitioni ng' reaction scheme for the Aplysia in enzyme, similar to that established previously for mammalian CD38/NAD(+) glycohydrolases. This mechanism involv es the formation of a single oxocarbenium-type intermediate that partitions to cADP-ribose and solvolytic products via competing pathways. In favour o f this mechanism was the finding that the enzyme also catalysed the hydroly sis of NMN+, a substrate that cannot undergo cyclization. The major differe nce between the mammalian and the invertebrate enzymes resides in their rel ative cyclization/hydrolysis rate-constant ratios, which dictate their resp ective yields of cADP-ribose (ADP-ribosyl cyclase activity) and ADP-ribose (NAD(+) glycohydrolase activity). For the Aplysia enzyme's catalysed transf ormation of NAD(+) we favour a mechanism where the formation of cADP-ribose precedes that of ADP-ribose; i.e. macroscopically the invertebrate ADP-rib osyl cyclase conforms to a sequential reaction pathway as a limiting form o f the partitioning mechanism.