摘要
Alkaline phosphatase is a stable enzyme which is strongly resistant to urea, guanidine hydrochloride, acid pH, and heat. But there have been few studies on the effect of organic cosolvents on the activity and structure of alkaline phosphatase. The activity of calf intestinal alkaline phosphatase (CIAP) is markedly increased when incubated in solutions with elevated trifluoroethanol (TFE) concentrations. The activation is a time dependent course. There is a very fast phase in the activation kinetics in the mixing dead time (30 s) using convential methods. Further activation after the very fast phase follows biphasic kinetics. The structural basis of the activation has been monitored by intrinsic fluorescence and far ultraviolet circular dichroism. TFE (060%) did not lead to any significant change in the intrinsic fluorescence emission maximum, indicating no significant change in the tertiary structure of CIAP. But TFE did significantly change the secondary structure of CIAP, especially increasing α helix content. We conclude that the activation of CIAP is due to its secondary structural change. The time for the secondary structure change induced by TFE preceds that of the activity increase. These results suggest that a rapid conformational change of CIAP induced by TFE results in the enhancement of CIAP activity, followed by further increase of this activity because of the further slightly slower rearrangements of the activated conformation. It is concluded that the higher catalytic activity of CIAP can be attained with various secondary structures.
Alkaline phosphatase is a stable enzyme which is strongly resistant to urea, guanidine hydrochloride, acid pH, and heat. But there have been few studies on the effect of organic cosolvents on the activity and structure of alkaline phosphatase. The activity of calf intestinal alkaline phosphatase (CIAP) is markedly increased when incubated in solutions with elevated trifluoroethanol (TFE) concentrations. The activation is a time dependent course. There is a very fast phase in the activation kinetics in the mixing dead time (30 s) using convential methods. Further activation after the very fast phase follows biphasic kinetics. The structural basis of the activation has been monitored by intrinsic fluorescence and far ultraviolet circular dichroism. TFE (060%) did not lead to any significant change in the intrinsic fluorescence emission maximum, indicating no significant change in the tertiary structure of CIAP. But TFE did significantly change the secondary structure of CIAP, especially increasing α helix content. We conclude that the activation of CIAP is due to its secondary structural change. The time for the secondary structure change induced by TFE preceds that of the activity increase. These results suggest that a rapid conformational change of CIAP induced by TFE results in the enhancement of CIAP activity, followed by further increase of this activity because of the further slightly slower rearrangements of the activated conformation. It is concluded that the higher catalytic activity of CIAP can be attained with various secondary structures.
基金
Supported by the National Key Basic Research Specific Foundation of China( No.G19990 75 60 7)
