Laboratory Exercises Measurement of k on Without a Rapid-Mixing Device* s

SUMMARY The slow, tight binding of bestatin to aminopeptidasefrom Aeromonas proteolytica is an uncommon biochemi-cal phenomenon that has enabled us to design a simple,inexpensive laboratory exercise for the measurement ofk on . The enzyme has good thermal stability and is toler-ant of freeze–thaw cycles, making it suitable for class-room experiments. The system is amenable to furtheraddition of experiments to an advanced biochemical orbiophysical laboratory class, including the measurementof k off , steady-state experiments with different inhibitors,and computer molecular modeling exploration of thecrystallographic structures of the aminopeptidase. Acknowledgments—The authors wish to thank Stephen Millsfor many helpful conversations in the design of this laboratory,Helene Citeau for assistance and maintenance of the instrumen-tation used in these experiments, and Sharon Ferguson for as-sistance in preparation of solutions for the experiment.REFERENCES [1] S. H. Wilkes, J. M. Prescott (1985) The slow, tight binding of bestatinand amastatin to aminopeptidases, J. Biol. Chem. 260,13154–13162.[2] B. Chevrier, C. Schalk, H. D’Orchymont, J. M. Rondeau, D. Moras,C. Tarnus (1994) Crystal structure of Aeromonas proteolytica amino-peptidase: A prototypical member of the co-catalytic zinc enzymefamily, Structure 2,283–291.[3] W. Desmarais, D. L. Bienvenue, K. P. Bzymek, G. A. Petsko, D.Ringe, R. C. Holz (2006) The high-resolution structures of the neutraland the low pH crystals of aminopeptidase from Aeromonas proteo-lytica, J. Biol. Inorg. Chem. 11,398–408.[4] C. C. Stamper, D. L. Bienvenue, B. Bennett, D. Ringe, G. A. Petsko,R. C. Holz (2004) Spectroscopic and X-ray crystallographic charac-terization of bestatin bound to the aminopeptidase from Aeromonas(Vibrio) proteolytica, Biochemistry 43,9620–9628.[5] C. Stamper, B. Bennett, T. Edwards, R. C. Holz, D. Ringe, G. Petsko(2001) Inhibition of the aminopeptidase from Aeromonas proteolyticaby L-leucinephosphonic acid. Spectroscopic and crystallographiccharacterization of the transition state of peptide hydrolysis, Bio-chemistry 40, 7035–7046.