Different catalytic properties of two highly homologous triosephosphate isomerase monomers

Abstract

It is assumed that amino acid sequence differences in highly homologous enzymes would be found at the peripheral level, subtle changes that would not necessarily affect catalysis. Here, we demonstrate that, using the same set of mutations at the level of the interface loop 3, the activity of a triosephosphate isomerase monomeric enzyme is ten times higher than that of a homologous enzyme with 74% identity and 86% similarity, whereas the activity of the native, dimeric enzymes is essentially the same. This is an example of how the dimeric biological unit evolved to compensate for the intrinsic differences found at the monomeric species level. Biophysical techniques of size exclusion chromatography, dynamic light scattering, X-ray crystallography, fluorescence and circular dichroism, as well as denaturation/renaturation assays with guanidinium hydrochloride and ANS binding, allowed us to fully characterize the properties of the new monomer.

Introduction

Sequence differences, both at DNA and protein level, are part of the basis of evolutionary change. Even in naturally occurring highly preserved proteins belonging to closely related species, there are different regions that normally do not influence on major structural or functional change. The highly preserved homodimeric glycolytic enzyme triosephosphate isomerase (TIM) has been well characterized over the last 50 years. Especially TIM’s from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), which share a similar amino acid sequence identity of 74% and 86%. However, it has been reported that, despite this, differences exist at the level of sensibility to thiol reagents [1], [2] and subtilisin [3], equilibrium unfolding [4], [5], as well as in the ability to form active dimers from unfolded monomers [6].

This paper demonstrates that variations in the sequence can also be related to significant catalytic differences of the TIM system. For this, a monomeric variant of TcTIM (monoTcTIM) was characterized. The monomeric nature of monoTcTIM was demonstrated using size exclusion chromatography, dynamic light scattering, by the fact that the activity is not dependent on the concentration and because the observed monophasic behavior in the denaturation/renaturation experiments. Although the stability of the monomeric enzyme was about 15% of that of the native dimer and an increase in ANS binding was observed, the overall three dimensional structure was preserved as shown using circular dichroism (CD), intrinsic fluorescence (IF) and X-ray crystallography experiments. It was found that the catalytic turnover is ten times higher than that for monoTbTIM [7], the equivalent monomeric mutant from T. brucei. Our results are discussed in light of the current information available for single mutants.