| In this thesis, I will present work pertaining to various aspects critical to the catalytic function of the enzyme, adenylate kinase (ADK), a nucleotide phosphoryl transferase.;The on-enzyme nucleotide equilibrium constant, Knuc, between the productive nucleotide ternary complexes, EMgADPADP and EMgATPAMP , was determined for E.coli ADK (EADK) by 31P-NMR to be 9.6 +/- 0.7 towards EMgADPADP . In conjunction with the maximum apparent rates of enzyme turnover determined in the steady-state, the microscopic rates of lid-opening from the EMgADPADP and EMgATPAMP complexes were determined to be 660 +/- 70 s-1 and 1900 +/- 160 s-1, respectively.;The role of the magnesium co-factor in the EADK phosphoryl transfer reaction was examined by measuring the magnesium dependence of the rates of enzyme turnover and NMR CPMG relaxation dispersion. Surprisingly, magnesium was found to directly accelerate the rate of the lid-opening conformational transition and, correspondingly, the rate of enzyme turnover. The role of magnesium in phosphoryl transfer could not be determined; however, using the on-enzyme nucleotide equilibrium constant, Knuc, the microscopic rates of phosphoryl transfer for WT EADK in the absence of magnesium were determined to be 250 +/- 30 s-1 and 2350 +/- 90 s -1 for the 2ADP→ATP+AMP and ATP+AMP→2ADP reactions, respectively. This may be the first time that the rates of ADK catalyzed phosphoryl transfer have been directly measured.;The lid-opening pathways of EADK and a homologous ADK (PADK) from P.profundum, a pressure tolerant bacteria isolated from deep-sea sediment, were explored by simulating the opening pathway by targeted molecular dynamics (TMD) and measuring rate of enzyme turnover as a function of pressure. The opening activation volumes were determined to be -33 +/- 5 mL/mol and 0 +/- 1.6 mL/mol for PADK and EADK, respectively. Using a combined approach of mutagenesis and targeted molecular dynamics (TMD) simulations, the decreased activation volume of PADK relative to EADK was linked to increased charge solvation in the lid-opening transition state ensemble (TSE). Our results indicate that the ADK opening TSE lies towards the closed conformation.;Based upon the results presented here we believe a consistent mechanistic framework to describe ADK function has been developed. The challenge now is to use this framework to better understand the catalytic power of ADK. |
