Kinetic analysis of cyclic CMP-specific and multifunctional phosphodiesterases by quantitative positive-ion fast-atom bombardment mass spectrometry

Abstract

Two enzymes, cyclic CMP-specific phosphodiesterase and multifunctional phosphodiesterase, are responsible for the hydrolysis of cytidine 3',5'-cyclic monophosphate in living cells. Quantitation of both enzymes has been carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubates after termination of the reaction. The kinetic data obtained are in close agreement with parallel data obtained by the conventional radiometric assay. The extra facility of the mass spectrometry based assay to monitor several incubation components simultaneously has been exploited to study the concurrent hydrolysis of alternate cyclic nucleotide substrates and provides kinetic parameters of significance in interpreting substrate-enzyme interactions. This is extended by the use of collisionally-induced dissociation of the protonated molecules of the liberated products to identify the mononucleotide isomers resulting from the cyclic nucleotide hydrolysis.

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

“…Additionally, the chitobiose reaction was allowed to proceed for 10 min compared to 4 min for chitotriose, chitotetraose, and chitopentaose, since chitobiose reacts much slower than the other three acceptor substrates. For the individual measurements of each chitooligosaccharide, PAPS was kept at a fixed, saturating concentration of 25 M. An average single-point normalization factor was determined to be 2.4 for chitobiose-6-OSO 3 Ϫ , 3.0 for chitotriose-6-OSO 3 Ϫ , 4.0 for chitotetraose-6-OSO 3 Ϫ , and 2.7 for chitopentaose-6-OSO 3 Ϫ . The initial reaction velocity was calculated and plotted versus the chitooligosaccharide concentration, generating four saturation plots for the four acceptor substrates (Figure 1) (Figure 2a, b, c, d).…”

“…The development of soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption (MALDI), has made mass spectrometry an excellent complementary technique to conventional spectrophotometric methods for studying enzyme kinetics [23][24][25][26][27][28][29]. With the improvements of resolving power, sensitivity, and versatility, mass spectrometry has become a highly competitive analytical method for detection and characterization of biochemical reaction intermediates that can be used to elucidate enzymatic reaction pathways and catalytic mechanisms [16 -22].…”

PDE7A1 hydrolyzes cCMP

“…Both PDE7A1 and MRP5 represent low-affinity and high-capacity mechanisms for the removal of cCMP, which additionally supports a potential function in the detoxification of exceedingly high cCMP concentrations. cCMP-degrading activity has been described in several types of cells and tissues [13][14][15][16][17][18][19][20][21] and was attributed to a ''cCMP-specific PDE'' and a ''multifunctional PDE'' (Suppl. Table 2).…”

“…Similarly, although the ''cCMP-specific PDE'' has K M values in the millimolar range that are very similar for both cAMP and cCMP (Suppl. Table 2), its ''specificity'' for cCMP is caused by the $200-fold higher V max value for cCMP hydrolysis, as compared to cAMP degradation [16].…”

“…A so-called ''cCMP-specific'' phosphodiesterase (PDE) was claimed with K M values in the millimolar range [13][14][15][16]. Moreover, a ''multifunctional cCMP-PDE'' was proposed with K M values <200 lM and activity for both 3 0 ,5 0 -and 2 0 3 0 -cNMPs [16][17][18][19][20][21]. However, the identity of these proteins is still elusive.…”