Abstract
Adenosine 5'-phosphate was synthesized with 3H or 14C label specifically located as [1'-3H]AMP, [1'-14C] AMP, [5'-3H]AMP, and [5'-14C]AMP. The synthesis was accomplished from adenine and glucose or adenine and ribose using enzymes from the pentose pathway and/or from the purine salvage pathways. Structural analysis of the compounds confirmed the locations of the radiolabels. The methods provide a general scheme for the efficient synthesis of adenine nucleotides of high purity with 3H or 14C at any stable position on the ribose ring. Synthesis of [5'-14C]dAMP and [1'-3H] dAMP from the corresponding ribonucleotides was accomplished with ribonucleotide reductase. Labeled inosine was prepared by enzymatic dephosphorylation and deamination of labeled AMP. These compounds have been used to measure the secondary kinetic isotope effects on the acid-catalyzed hydrolysis of the N-glycosidic bond of AMP, dAMP, and inosine and the corresponding primary kinetic isotope effects with AMP. Acid hydrolysis in 0.1 or 0.2 N HCl at 50 degrees C gave 1H/3H secondary kinetic isotope effects of 1.23 +/- 0.01, 1.26 +/- 0.01, and 1.230 +/- 0.003 for AMP, dAMP, and inosine, respectively. The primary kinetic isotope effect for 12C/14C was 1.049 +/- 0.010 for AMP. The apparent rate constants for hydrolysis under these conditions were similar for inosine and AMP and were in the range 10(-6)-10(-5)s-1. Acid hydrolysis of dAMP is approximately 1000-fold faster than AMP but gives a similar 1H/3H kinetic isotope effect. The results of secondary isotope effects indicate that the transition states for the acid-catalyzed hydrolysis of the N-glycosidic bonds of inosine, AMP, and dAMP have similar bonding to 1'-3H in the transition state and have considerable carboxonium character. Results with [1'-14C]AMP demonstrate that a significant primary isotope effect can be measured in the acid solvolysis of the N-glycosidic bond of AMP.
Highlights
Adenosine S'-phosphate was synthesized with 'H or 14Clabel located as [l'-'H]AMP, [1'-l4C] AMP, [5'-'H]AMP, and [5'-14C]AMP
The values reported here are consistent with the previous proposals that acidcatalyzed hydrolysis of purine nucleosides [2,3,4] and nucleotides [5]proceed through transition states with considerable carboxonium character
We present procedures for the enzymatic synthesis of adenine nucleotides of high purity and with specific labels of 3Hand "C in the ribose ring
Summary
Adenosine S'-phosphate was synthesized with 'H or 14Clabel located as [l'-'H]AMP, [1'-l4C] AMP, [5'-'H]AMP, and [5'-14C]AMP. Labeled inosine was prepared by enzymatic dephosphorylation and deamination of labeled AMP These compounds have been used to measure the secondary kinetic isotope effects on the acid-catalyzed hydrolysis of the N-glycosidic bond of AMP, dAMP, and inosine and the corresponding primary kinetic isotope effects with AMP. The values reported here are consistent with the previous proposals that acidcatalyzed hydrolysis of purine nucleosides [2,3,4] and nucleotides [5]proceed through transition states with considerable carboxonium character The results of these studies provide values which can be compared to the kinetic isotope effects for AMP nucleosidases which catalyze the same hydrolytic reaction with AMP, but with a rate enhancement of approximately 10" [6]. The methods provided here can be applied to the specific labeling of a wide variety of both purine and pyrimidine nucleotides at any position of the ribose rings and should prove useful for kinetic isotope studies with other enzymes
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