Protein Engineering, Vol. 12, No. 3, 243-250,
March 1999
© 1999 Oxford University Press
Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power
European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 102209, D-69012 Heidelberg, Germany, 3 Vrije Universiteit Brussel, Instituut voor Moleculaire Biologie, Paardenstraat 65, B-1640 Sint-Genesius-Rode, 4 Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Catholic University of Louvain, Avenue Hippocrate 74, B-1200 Brussels, 5 Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2620 Wilrijk, Belgium and 6 Department of Biochemistry, University of Oulu, Linnanmaa, FIN-90571 Oulu, Finland
The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 Å crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26°C (57°C for leishmania TIM to 83°C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.
Keywords: enzymology/leishmania/stability/structure/triosephosphate isomerase
1 Present address: Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY10032, USA
2 Present address: Max Planck Institut für Biophysik,Heinrich Hoffmann Str. 7, D-60528 Frankfurt, Germany
7 To whom correspondence should be addressed. E-mail: rik.wierenga{at}oulu.fi
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