A quenched-flow system for measuring heterogeneous enzyme kinetics with sub-second time resolution.

Area of Science:

• Biochemistry
• Enzymology
• Chemical Kinetics

Background:

• Enzyme reactions at insoluble substrates are understudied compared to homogeneous reactions.
• Interfacial enzyme kinetics are complex, involving multiple steps.
• Traditional pre-steady-state kinetics methods are often unsuitable for precipitating substrates.

Purpose of the Study:

• To develop and validate a quenched-flow system for transient kinetics of heterogeneous enzyme reactions.
• To investigate the distinct mechanisms of endo-lytic and exo-lytic cellulases on insoluble substrates.

Main Methods:

• A novel quenched-flow system utilizing peristaltic pumps and stirred substrate suspensions.
• Achieved a dead time below 100 milliseconds for high time resolution.
• Validated with alkali-catalyzed hydrolysis of 2,4-dinitrophenyl acetate (DNPA).
• Applied to study cellulases Cel7A and Cel7B acting on microcrystalline cellulose (Avicel).

Main Results:

• The system successfully measured transient kinetics for heterogeneous reactions.
• Endo-lytic Cel7B exhibited rapid substrate binding and maximal activity within the instrument's dead time.
• Exo-lytic Cel7A demonstrated slower initiation, reaching maximal activity after 5-8 seconds with a 10-fold lower turnover rate.
• Distinct kinetic profiles were observed for the two cellulases.

Conclusions:

• The developed quenched-flow system is effective for studying enzyme mechanisms on insoluble substrates.
• Significant kinetic differences exist between endo- and exo-lytic cellulase mechanisms.
• This instrument offers a valuable tool for elucidating interfacial enzyme kinetics.