The pH and temperature dependence of the activity of the high Km cyclic nucleotide phosphodiesterase of bakers' yeast.
The hydrolysis of adenosine 3':5'-monophosphate by the high Km cyclic nucleotide phosphodiesterase of bakers' yeast was studied over a range of temperature and pH at I = 0.17. The effects of ionic strength and MgCl2 concentration were studied at pH 7.7 and 30 degrees C. Km and Vmax were insensitive to changes in the MgCl2 concentration between 1 and 30 mM, implying that this enzyme (which does not require free divalent metal ions) does not discriminate between free cyclic AMP- and the Mg-cyclic AMP+ complex. Vmax decreased below pH 6.8 because of protonation of a group required in the basic form in the enzyme x substrate complex. On the basis of its pK (5.46 at 30 degrees C) and delta H (23 kJ/mol) this group was tentatively identified as imidazole. Vmax/Km decreased above pH 6.8 because of ionization of a group required in the acid form in the free enzyme, with a pK of 7.88 at 30 degrees C and a delta H of about 13 kJ/mol. Several possibilities exist for the identity of this group, the most likely being a second imidazole, sulfhydryl, or a water molecule bonded to tightly bound zinc. At pH 7.90, log Vmax and log Km both changed linearly with 1/T (between 12 degrees C and 37 degrees C) with enthalpies of 47 and 55 kJ/mol, respectively. Consequently, at low enough cyclic AMP concentration, the rate of reaction at pH 7.90 decreases slightly when the temperature is increased. This is also true at higher pH, but in the physiological pH range (6.4 to 7.5) Vmax/Km and, therefore, the rate of reaction at very low cyclic AMP concentration were nearly independent of temperature. Under physiological conditions, the Km approaches the upper limit of in vivo cyclic AMP concentrations in yeast, and at normal in vivo cyclic AMP concentrations the pH optimum is within or below the physiological range of pH in yeast.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008433:Mathematics; D011189:Potassium Chloride / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013696:Temperature
D015636:Magnesium Chloride; D011189:Potassium Chloride; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D008274:Magnesium