Transition State Analysis of SAMHD1 from Primary ¹⁸O, ³³P, and Solvent Kinetic Isotope Effects

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Human sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) is a key enzyme regulating deoxynucleotide pools and DNA repair.
• Understanding the catalytic mechanism of SAMHD1, particularly its transition state, is crucial for elucidating its biological roles.

Purpose of the Study:

• To characterize the chemical features of the SAMHD1 transition state during 2'-deoxyadenosine 5'-triphosphate (dATP) hydrolysis.
• To elucidate the mechanistic details of phosphohydrolase activity using primary kinetic isotope effects and solvent isotope effects.

Main Methods:

• Analysis of 18O and 33P primary kinetic isotope effects (KIEs) at the α-phosphoryl group of dATP.
• Measurement of solvent 2H2O isotope effects for dATP hydrolysis.
• Quantum chemical calculations to model the transition state structure.

Main Results:

• Intrinsic KIE values for [5'-18O]dATP (1.028 ± 0.003) and [α-33P]dATP (1.015 ± 0.004) were determined.
• A solvent KIE of 3.2 ± 0.1 indicated single proton transfer at the transition state.
• Quantum chemical matching supported a concerted, loose, highly asymmetric D N A N transition state.

Conclusions:

• The study provides the first detailed characterization of a triphosphohydrolase transition state.
• The findings reveal a near-midpoint proton transfer from His215 to the deoxyadenosine 5'-oxygen.
• This work demonstrates the utility of 33P primary isotope effects for characterizing phosphotransferase transition states.