Influence of the mutation topology on
properties of human
cystathionine beta-synthase (CBS) polypeptide
M. Janosik1, K. Jelinek2, P. Melenovska1, V. Klatovska1, J. Krijt1 and V. Kozich1
1Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague,
Ke Karlovu 2, 12000 Prague 2, Czech Republic
2Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Hlavova 2030, 12843 Prague 2, Czech Republic
Keywords: homocystinuria, cystathionine beta-synthase, mutation topology, misfolding
Cystathionine beta-synthase, a key enzyme of the methionine cycle, catalyzes condensation of toxic homocysteine and serine to cystathionine. More than 140 different mutations have been found. However only a small proportion has been characterized in more detail.
In this study we have classified topology of 27 known mutations based on the CBS active core structure and the structure of CBS domains in various proteins. Selected mutations represent disturbances in various protein domains and cover about one fifth of all mutations found in CBS patients. Further we expressed all 27 mutants in E.coli and Chinese hamster ovary cells expression systems. We measured the catalytic activity and monitored the degree of assembly by native western blotting. We correlate tetramer amounts and activity with topology of CBS mutations.
Our data suggest that a substantial proportion of selected mutations have an internal propensity to misfold and aggregate. The properties of the mutant enzyme is significantly affected by the location of the mutation. Mutations buried in CBS globule cause more severe disturbances of quaternary enzyme structure and activity than the mutations at the surface of the enzyme.
This work is being supported by the Wellcome Trust grant 070255/Z03/Z.