Determining Optimum Temperature and Ph for Enzymatic Reactions of Alpha Amylase
Biochem. J. (1995) 305, 17-20 (Printed in Great Britain)
17
RESEARCH COMMUNICATION
The effect of low temperatures
Nicole MORE, Roy M. DANIEL* and Helen H. PETACH
on enzyme
activity
Thermophile Research Unit, University of Waikato, Private Bag 3105, Hamilton 2001, New Zealand
The stability of two enzymes from extreme thermophiles (glutamate dehydrogenase from Thermococcales strain ANI and f,-
enzymes,
glucosidase from Caldocellum saccharolyticum expressed in Escherichia coli) has been exploited to allow measurement of activity over a 175 °C temperature range, from + 90°C to -85 °C for the glutamate dehydrogenase and from + 90 °C to -70°C for the ,-glucosidase. The Arrhenius plots of these
and those for two mesophilic enzymes (glutamate dehydrogenase from bovine liver and ,)-galactosidase from Escherichia coli), exhibit no downward deflection corresponding to the glass transition, found by biophysical measurements of several non-enzymic mesophilic proteins at about -65 °C and reflecting a sharp decrease in protein flexibility as the overall motion of groups of atoms ceases.
INTRODUCTION
It is now accepted that, in general terms, enzyme activity is dependent on protein dynamics [1-5]: in other words, an enzyme must be flexible to function. A number of biophysical studies [6-12] have shown that the flexibility of mesophilic proteins undergoes a transition at about -65 °C. Below this transition temperature the overall motion of groups of atoms within the protein ceases, and all that is left is the harmonic vibration of individual atoms. These results suggest that protein function will also cease at this temperature. The experiments which come closest to demonstrating this are those of Petsko's group [13], which show that, above the transition temperature, crystals of RNAase A rapidly bind the inhibitor cytidine 2'-monophosphate, but at -61 °C do not. The transition is sharp and is manifested by distinct changes in slope. Furthermore,
17
RESEARCH COMMUNICATION
The effect of low temperatures
Nicole MORE, Roy M. DANIEL* and Helen H. PETACH
on enzyme
activity
Thermophile Research Unit, University of Waikato, Private Bag 3105, Hamilton 2001, New Zealand
The stability of two enzymes from extreme thermophiles (glutamate dehydrogenase from Thermococcales strain ANI and f,-
enzymes,
glucosidase from Caldocellum saccharolyticum expressed in Escherichia coli) has been exploited to allow measurement of activity over a 175 °C temperature range, from + 90°C to -85 °C for the glutamate dehydrogenase and from + 90 °C to -70°C for the ,-glucosidase. The Arrhenius plots of these
and those for two mesophilic enzymes (glutamate dehydrogenase from bovine liver and ,)-galactosidase from Escherichia coli), exhibit no downward deflection corresponding to the glass transition, found by biophysical measurements of several non-enzymic mesophilic proteins at about -65 °C and reflecting a sharp decrease in protein flexibility as the overall motion of groups of atoms ceases.
INTRODUCTION
It is now accepted that, in general terms, enzyme activity is dependent on protein dynamics [1-5]: in other words, an enzyme must be flexible to function. A number of biophysical studies [6-12] have shown that the flexibility of mesophilic proteins undergoes a transition at about -65 °C. Below this transition temperature the overall motion of groups of atoms within the protein ceases, and all that is left is the harmonic vibration of individual atoms. These results suggest that protein function will also cease at this temperature. The experiments which come closest to demonstrating this are those of Petsko's group [13], which show that, above the transition temperature, crystals of RNAase A rapidly bind the inhibitor cytidine 2'-monophosphate, but at -61 °C do not. The transition is sharp and is manifested by distinct changes in slope. Furthermore,