Roozbeh Eskandari, PhD
Albert Einstein College of Medicine
Oxocarbenium-ion Character of Human DNPH1 Guides Transition State Analogs Design
Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1 (DNPH1) controls the level of hydroxymethyl deoxyuridine monophosphate (hmdUMP), and its action attenuates the level of incorporation of the modified nucleotide into DNA. DNPH1 catalyzes a relatively fast hydrolytic depyrimidination (kcat = 0.33 s-1) of 5-hydroxymethyl deoxyuridine, yielding hmUracil (hmU) and 2-deoxyribose-5-phosphtae (2dR5R). The reaction is catalyzed by a double-displacement mechanism with a covalent intermediate. Analysis of the transition state of the first chemical step using multiple kinetic isotope effect (KIE) measurements revealed a dissociative mechanism with a higher energetic barrier to form 2-deoxyribocation followed by nucleophilic attack by glutamate residue (E104). Consequently, a relatively faster nucleophilic attack of a water molecule on the glycosyl-enzyme covalent intermediate releases 2dR5P. An investigation through density functional theory to match the intrinsic KIEs suggests that the bond distance at the transition state between E104 and the anomeric carbon (rC-O) is 2.3. These results confirm a dissociative DN‡*AN transition state with complete hmU loss and partially dissociative nucleophilic glutamate residue (E104). The transition state model predicts that deoxyribose adopts a 3ʹ-exo puckering during nucleophilic attack. The information on reaction mechanisms helped us design inhibitors with an inhibition constant lower than 10 nM.
Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1 (DNPH1) controls the level of hydroxymethyl deoxyuridine monophosphate (hmdUMP), and its action attenuates the level of incorporation of the modified nucleotide into DNA. DNPH1 catalyzes a relatively fast hydrolytic depyrimidination (kcat = 0.33 s-1) of 5-hydroxymethyl deoxyuridine, yielding hmUracil (hmU) and 2-deoxyribose-5-phosphtae (2dR5R). The reaction is catalyzed by a double-displacement mechanism with a covalent intermediate. Analysis of the transition state of the first chemical step using multiple kinetic isotope effect (KIE) measurements revealed a dissociative mechanism with a higher energetic barrier to form 2-deoxyribocation followed by nucleophilic attack by glutamate residue (E104). Consequently, a relatively faster nucleophilic attack of a water molecule on the glycosyl-enzyme covalent intermediate releases 2dR5P. An investigation through density functional theory to match the intrinsic KIEs suggests that the bond distance at the transition state between E104 and the anomeric carbon (rC-O) is 2.3. These results confirm a dissociative DN‡*AN transition state with complete hmU loss and partially dissociative nucleophilic glutamate residue (E104). The transition state model predicts that deoxyribose adopts a 3ʹ-exo puckering during nucleophilic attack. The information on reaction mechanisms helped us design inhibitors with an inhibition constant lower than 10 nM.
