Kinetic competence of the cADP-ribose-CD38 complex as an intermediate in the CD38/NAD(+) glycohydrolase-catalysed reactions: implication for CD38 signalling

CD38/NAD(+) glycohydrolase is a type II transmembrane glycoprotein widely u sed to study T- and B-cell activation and differentiation. CD38 is endowed with two different activities: it is a signal transduction molecule and an ectoenzyme that converts NAD(+) into ADP-ribose (NAD(+) glycohydrolase acti vity) and small proportions of cADP-ribose (cADPR; ADP-ribosyl cyclase acti vity), a calcium-mobilizing metabolite, which, ultimately, can also be hydr olysed (cADPR hydrolase activity). The relationship between these two prope rties, and strikingly the requirement for signalling in the formation of fr ee or enzyme-complexed cADPR, is still ill-defined. In the present study we wanted to test whether the CD38-cADPR complex is kinetically competent in the conversion of NAD(+) into the reaction product ADP-ribose. In principle , such a complex could be invoked for cross-talk, via conformational change s, with neighbouring partner(s) of CD38 thus triggering the signalling phen omena. Analysis of the kinetic parameters measured for the CD38/NAD(+) glyc ohydrolase-catalysed hydrolysis of 2'-deoxy-2'-aminoribo-NAD(+) and ADP-cyc lo[N1,C1']-2'-deoxy-2'-aminoribose (slowly hydrolysable analogues of NAD(+) and cADPR respectively) ruled out that the CD38-cADPR complex can accumula te under steady-state conditions. This was borne out by simulation of the p revalent kinetic mechanism of CD38, which involve the partitioning of a com mon E . ADP-ribosyl intermediate in the formation of the enzyme-catalysed r eaction products. Using this mechanism, microscopic rate conditions were fo und which transform a NAD(+) glycohydrolase into an ADP-ribosyl cyclase. Al together, the present work shows that if the cross-talk with a partner depe nds on a conformational change of CD38, this is most probably not attributa ble to the formation of the CD38-cADPR complex. In line with recent results on the conformational change triggered by CD38 ligands [Berthelier, Labour eau, Boulla, Schuber and Deterre (2000) Eur. J. Biochem. 267, 3056-3064], w e believe that the Michaelis CD38-NAD(+) complex could play such a role ins tead.