Temperature of Maximum Stability
The observed activity (i.e., ADP synthesis by means of a phosphoryl transfer from ATP to AMP) for a given variant was assessed as a function of temperature, permitting the identification of the temperature at which each enzyme has maximum activity (Tma; see Figure 4).
Briefly, diluted adenylate kinase (AdK) (5 nM) is incubated in base buffer (25 mM potassium phosphate, 5 mM magnesium chloride, 65 mM potassium chloride, pH 7.3) with 1 mM DTT and 2 mM ATP at the desired temperature for 5 min. Enzymatic turnover is initiated by the addition of AMP at a final concentration of 2 mM. Reaction is allowed to proceed for 8 min at the desired temperature before being quenched in a twofold dilution with an ice-cold inhibitor, P1,P5-di(adenosine-50) pentaphosphate (AP5A; 1 mM final concentration), and transferred to an ice water bath. The quenched AdK/AP5A sample is incubated for 5 min in an ice water bath before being transferred to a secondary PK/LDH reaction mixture (additional information is given in Supplemental Information).
Turnover of NADH by the secondary enzymes is used as a direct proxy for the adenosine diphosphate (ADP) synthesized by AdK during the eight minute incubation, prior to thermal and AP5A quenching.
Parameterization of the observed thermoactivity properties of each enzyme was made by fitting the temperature dependent change in 340 nm absorbance (Activity) to a skewed gaussian distribution (Rusch and Lelieur, 1973):
(see equations and details in SI)
Fits of the experimental data to the described function were made using a simple least squares minimization,