Abstract
Mutations, replacing amino acids involved in the formation of hydrogen bonds between subunits of dimeric alkaline phosphatase, have been introduced. Influence of mutations on kinetic properties and structural stability of mutant enzymes was established. In addition, alterations in protein dynamic proper- ties have been studied using room temperature phosphorescence. Kinetic properties of both mutant en- zymes were virtually the same, differing from the wild type enzyme in the kcat value that was almost twice lower. Changes in protein dynamic properties of mutant proteins, compared to the wild type enzyme, did not parallel changes in kinetic properties suggesting that an alteration in the rigidity of the Trp109 envi- ronment is not responsible for the reduction of kinetic properties. Instead, combined kinetic and dynamic consequences of introduced mutations suggest that breaking of specific links, involved in transmission of conformational change, could be responsible for altered kinetic properties. (doi: 10.5562/cca2168)
Highlights
Alkaline phosphatase (AP) from Escherichia coli (E.C. 3.1.3.1), is a dimeric metalloenzyme that catalyzes the hydrolysis of a wide variety of phosphomonoesters
Altered kinetic properties could be the consequence of a change in protein rigidity, or disruption of the bonding interaction involved in transmission of conformational change between the active sites on neighboring subunits.[10]
It has been already shown that the T81A mutation, located at the subunit interface, influences the kinetic properties of AP
Summary
Alkaline phosphatase (AP) from Escherichia coli (E.C. 3.1.3.1), is a dimeric metalloenzyme that catalyzes the hydrolysis of a wide variety of phosphomonoesters. Consequences of additional weakening of the link between the β-sheets at the same site were probed by a new hydrogen bond removing mutation, created by substituting Gln[83] with Leu. Kinetic properties of single T81A and double T81A/Q83L mutant proteins and the wild-type enzyme have been compared in Tris/HCl (c(Tris) = 1 mol dm–3, pH = 8.0). Activity was measured in a reaction mixture, containing 2.3 cm[3] of Tris/HCl (c(Tris) = 1 mol dm–3, pH = 8.0), 0.100 cm[3] of enzyme solution in Tris/HCl (c(Tris) = 20 mmol dm–3, pH = 7.6), and 0.100 cm[3] of substrate solution (p-nitrophenyl phosphate hexahydrate, disodium salt; pNPP) of an appropriate concentration. The rate of inactivation was determined by fitting the experimental data to a single-exponential decay function
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