MECHANISTIC ANALYSIS OF THE CYSTINE LYASE C-DES FROM SYNECHOCYSTIS SP. PCC 6803: STABILITY OF THE a-AMINOACRYLATE INTERMEDIATE, FORMATION OF THIAZOLIDINE ADDUCT AND IDENTITY OF THE CATALYTIC BASE
Open Access
Author:
Koch, Gretchen
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
July 20, 2009
Committee Members:
Bollinger, Thesis Advisor/Co-Advisor Joseph M Bollinger Jr., Thesis Advisor/Co-Advisor
The open reading frame slr2143 from Synechocystis sp. PCC 6803 encodes the enzyme C-DES: a pyridoxal phosphate enzyme that catalyzes the b-elimination of L-cystine to produce activated persulfide, pyruvate and ammonia. The mechanism of this reaction was characterized in both the transient and steady state kinetic regimes in order to better understand the intricacies in this reaction compared to the better understood cysteine desulfurase reaction. Two kinetically separated species form with absorbancies in the 465-495 nm range that may be attributed to -aminoacrylate species. These intermediates were differentiated by a slight shift in the visible region and variable reactivity upon addition of L-cysteine or sodium azide, leading us to conclude that the slower forming intermediate is not a typical -aminoacrylate, as previously suggested.
Through stopped-flow spectroscopy and mass spectrometry, we can conclude that the lack of a nucleophilic cysteine residue in the active site of C-DES leads to a change in the rate determining step upon use of cysteine as a substrate instead of cystine. The lack of this nucleophilic attack means that a stable thiazolidine adduct forms before the deprotonation step, halting the reaction before productive -elimination can occur. This adduct formation explains the low baseline activity of C-DES with cysteine as a substrate and the spectral appearance of a stable, 350-nm-absorbing species. A protein variant replacing a candidate catalytic base, histidine 114, with alanine was also constructed and characterized. The activity of the variant is greatly reduced and the absorption spectra of the protein upon binding of substrate shifted in such a way that this histidine residue likely plays both a catalytic role and interacts with substrate upon its binding.