Allosteric activation of aspartate transcarbamylase with a fluorescent nucleotide analogue: linear-benzo-ATP.

P Vanderlijn,J.R Barrio,N.J Leonard

doi:10.1016/s0021-9258(17)34232-1

Abstract

The interaction of Escherichia coli aspartate transcarbamylase with linear-benzo-ATP has been investigated by means of fluorescence spectroscopy. The fluorescent nucleotide analogue activates the enzyme to the same extent as ATP. Fluorescence polarization has been used to determine the association constant of lin-benzo-ATP with aspartate transcarbamylase (ATCase) which is 5 X 10(-3) M-1 at pH 8.7, at 4 degrees C, assuming six binding sites. This association constant is similar to those previously obtained for ATP at a variety of temperatures, buffers, and pH. The fluorescence emission of lin-benzo-ATP is not quenched when bound to ATCase, which indicates absence of pi interactions between the activator and tyrosyl residues in the protein. These residues have been implicated in the stereochemical mechanism of allosteric interactions in ATCase. Furthermore, this fluorescence behavior implicates hydrogen bond formation between the amino group of lin-benzo-ATP and a nucleophilic center at the enzyme binding site. The fact that lin-benzo-ATP activates ATCase is consistent with a previously published model for nucleotide regulation of the enzyme.

Highlights

Has been investigated by means of fluorescence spectroscopy
Is not quenched when bound to ATCase, which indicates absence of rr interactions between the activator and tyrosyl residues in the protein. These residues have been implicated in the stereochemical mechanism of allosteric interactions in ATCase
-0 with the added feature that a benzene ring has been inserted between the imidazole and pyrimidine rings [4,5,6,7]. lin-Benzoadenine nucleotide analogues have previously been shown to be active with a wide variety of enzymes [6,7,8,9,10] and to possess spectroscopic properties which allow their use as microenvironmental probes of adenine binding sites (1 1).2* ” In this paper, we utilize the fluorescence properties of Zin-benzo-ATP (I) to obtain information about the nature and type of binding of nucleotides to the allosteric site of ATCase

Summary

Introduction

Has been investigated by means of fluorescence spectroscopy. The fluorescent nucleotide analogue activates the enzyme to the same extent as ATP. Lin-Benzoadenine nucleotide analogues have previously been shown to be active with a wide variety of enzymes [6,7,8,9,10] and to possess spectroscopic properties which allow their use as microenvironmental probes of adenine binding sites (1 1).2* ” In this paper, we utilize the fluorescence properties of Zin-benzo-ATP (I) to obtain information about the nature and type of binding of nucleotides to the allosteric site of ATCase. J. for linear-Benzo-ATP: An Allosteric Activator of ATCase the treatment of the polarization data and the calculation of the binding curve were aided by the use of a Monroe 1880 programmable calculator.

Results

Conclusion