A Fluorescent Probe-labeled Escherichia coli Aspartate Transcarbamoylase That Monitors the Allosteric Conformational State

Hiro Tsuruta,Evan R. Kantrowitz,Jay M. West

doi:10.1074/jbc.m304018200

Abstract

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.

Highlights

Allosteric regulation of enzymatic activity is manifested by the ability of the enzyme to exist in at least two different structural and functional forms [1]
To be able to observe the actual T to R transition in aspartate transcarbamoylase, as well as to monitor structural changes induced by the heterotropic effectors, we have developed a fluorescent-labeled version of aspartate transcarbamoylase that has kinetic characteristics similar to the wild-type enzyme, and fluoresces uniquely in the R quaternary structure
The fluorescent label was attached to the enzyme site- in the 240s loop of the catalytic chain of the enzyme because the 240s loops at the C1:C4 interface are much closer in the R state than in the T state [13, 24]

Summary

Introduction

Allosteric regulation of enzymatic activity is manifested by the ability of the enzyme to exist in at least two different structural and functional forms [1]. Fluorescent Pyrene-labeled Aspartate Transcarbamoylase quaternary structural changes, several tertiary changes occur during the T to R state transition. To create a fluorescent-labeled enzyme, which could monitor the allosteric transition, we conjugated a fluorophore site via a Cys residue at position 241 of the 240s loop (Fig. 1). Pyrene site attached to multiple sites on a protein has been utilized in a number of studies as a “molecular ruler” with a range of ϳ0 –20 Å It is capable of sensitively monitoring a conformational change in proteins [22, 23]. We used the pyrene-labeled aspartate transcarbamoylase to monitor the allosteric transition and the movement of the 240s loop into the R state conformation, induced by the binding of substrates, substrate analogues, and allosteric effectors

Methods

Results

Conclusion