ANALYTICAL BIOCHEMISTRY 237, 260±273 (1996) ARTICLE NO. 0238 Program DYNAFIT for the Analysis of Enzyme Kinetic Data: Application to HIV Proteinase

factory ®t of more than one progress curve to the same A computer program with the code name DYNAFIT set of rate constants, and it can be used for active-site was developed for ®tting either the initial velocities titrations. Finally, DYNAFIT can treat as adjustable pa-or the time course of enzyme reactions to an arbitrary rameters certain instrumental parameters, such as base-molecular mechanism represented symbolically by a line signal or molar absorptivity. set of chemical equations. Seven numerical tests and Classical enzyme kinetics (3±5) is concerned with ®ve graphical tests are applied to judge the goodness ®tting a set experimental data to a single algebraic of ®t. Experimental data on the inhibition of the dis- equation, namely the exact or analytical rate equation sociative dimeric proteinase from HIV were used in based on the postulated reaction mechanism. The goal four test examples. A set of initial velocities was ana- is to determine the value of certain kinetic constants lyzed to see if a tight-binding inhibitor could bind to or, more importantly, to see if the mechanism ®ts thethe HIV proteinase monomer. Three different sets of data. For many important mechanisms, such as tight-progress curves were analyzed (i) to determine the ki-binding inhibition of dissociative dimeric enzymesnetic properties of an irreversible inhibitor, (ii) to in-(retroviral proteinases, integrases, and reverse tran-vestigate the dissociation and denaturation mecha-scriptases), the exact rate equation unfortunately can-nism for the protease dimer, and (iii) to investigate the not be derived. In order to determine the inhibitioninhibition mechanism for a transient inhibitor. The constants or validate a postulated inhibition mecha-program is available by anonymous ftp via uwmml.ph nism, one must resort to an inexact or ®nite-differencearmacy.wisc.edu and on the World Wide Web vi