Structural and functional analysis of cysteine residues in human glutamate decarboxylase 65 (GAD65) and GAD67.
Previously, we have shown that brain glutamate decarboxylase (GAD) is greatly inhibited by sulfhydryl reactive reagent suggesting cysteine residue(s) may play an important role in GAD function. In this report, we determined the role of cysteine residues in the recombinant human 65-kDa GAD isoform (hGAD65) and 67-kDa GAD isoform (hGAD67), using a combination of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and site-directed mutagenesis. Here, we report that cysteine 446 (C446) in hGAD65 is important for its activity and is present as free sulfhydryl group. This conclusion is based on the following observations: (i) mutation of C446 in hGAD65 to alanine reduced hGAD65 activity by more than 90%, (ii) MALDI-TOF analysis of the non-reduced, trypsin-digested GAD65 revealed that C446 is present as a free sulfhydryl group as indicated by a peak at m/z (mass/charge) 647.3446 (peptide 443-448) and, when GAD65 was treated with sulfhydryl reagent, N-ethylmaleimide (NEM), the peak is shifted to m/z 772.3702,a mass increase of 125.1 daltons (Da) as a result of modification of cysteine by NEM. Parallel studies have also been conducted with hGAD67. Cysteine 455 was found to be important for GAD67 activity.
Wei
Jianning
J
Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, Florida 33431, USA.
Wu
Jang-Yen
JY
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2; D003545:Cysteine