Surface mapping of cystathione beta synthase: insigth into enzyme autoinhibition using mass spectrometry

 

Hnízda A.,Kožich V.

 

Institute of Inherited Metabolic Disorders, Charles University in Prague, First Faculty of Medicine

ales.hnizda@lf1.cuni.cz

 

Cystathionine β-synthase (CBS) is a tetrameric enzyme containing 551 amino acids, which catalyzes condensation of serine with homocysteine. Sequence of CBS can be divided to three regions: N-terminal part (1-39), active core (40-413) and C-terminal part (414-551); interaction of active core with C-termain domain causes enzyme autoinhibition.

 The 3-D structure was determined only for the truncated CBS lacking C-terminal part (amino acids 1-413, trCBS) since full-length CBS protein (wtCBS) could not be succesfully crystallized. The aim of this work is to describe molecular mechanism of the autoinhibition using the chemical modification of surface exposed amino acid residues followed by mass spectrometric detection.

Initially, we tested eight labelling compounds and six of them were suitable since they have not altered the quarternary structure and activity of CBS, namely 4 –hydroxyphenylglyoxal (HPG), N-ethylmaleinimide (NEM), diethylpyrocarbonate (DEP), N- hydroxysulfosuccinimide acetate (NHS), N-brom-succinimide (NBS) and N-acetylimidazole (NAI).

 In our ongoing study, we have analysed reactivity of four agents (NEM, DEP, NBS, NAI) with trCBS and wtCBS. Cluster of three tryptophane residues (Trp408-Trp410) was differentially reactive with NBS, modified in trCBS but not in wtCBS, indicating that the cluster is sterically hindered in wtCBS.

 Contradictory, cysteine (reacted with NEM), histidine (DEP) and tyrosine (NAI) modification sites were identically localized in both forms of CBS. These data shows subtle differences in surface of trCBS and wtCBS and the modular character of the enzyme. Futhermore, this dataset provides the restrains for computation modelling which would have explained the molecular mechanism of  CBS activity regulation.

 

This work was supported by Welcome Trust Internation Fellowship in Biomedical Science and by Research Project of Charles Universitz No. VZ MSM0021620806.