Holliday, Phillip (2009) An investigation of the response of a model class IIIA Adenyly Cyclase to carbon dioxide. Doctoral thesis, Durham University.
Adenylyl cyclase catalyses the conversion of adenosine triphosphate into 3’,5'-cyclic adenosine monophosphate and pyrophosphate. Adenylyl cyclases are grouped into six distinct Classes based on amino acid sequence similarity. Mammalian adenylyl cyclases and numerous prokaryotic adenylyl cyclases are grouped into Class III. Class III is further sub-divided into four sub-Classes, IIIa, IIIb, IIIc, and IIId, based upon amino acid polymorphisms within the active site. Class IIIa adenylyl cyclases include the mammalian G- protein regulated transmembrane adenylyl cyclases and numerous prokaryotic adenylyl cyclases such as Rv1625c from Mycobacterium tuberculosis. Class IIIb adenylyl cyclases include the mammalian soluble adenylyl cyclase and numerous prokaryotic adenylyl cyclase such as CyaB1 from Anabaena PCC 7120. Class IIIb adenylyl cyclases are stimulated by inorganic carbon. New findings dispute whether the inorganic carbon stimulation of Class IIIb adenylyl cyclases is by HCO(_3) or CO(_2). Class IIIa adenylyl cyclases have previously been demonstrated to be non-responsive to HCO(_3) but have not been investigated with CO(_2) .Experiments were performed using the prokaryotic Class IIIa adenylyl cyclase Rv1625c(_204-443)- The specific activity of Rvl625c (_204-443) was obtained by measuring the conversion of [a-(32)P]ATP into [(^32)P]cAMP. Rv1625c (_204-443) was assayed for pH dependence, cation dependence, dose dependence, enzyme kinetics, and inorganic carbon-activating species in the presence or absence of inorganic carbon. The results showed that in vitro Rv1625c(_204-443) is stimulated by CO(_2) and not HCO(_3) below pH 7.5 with an apparent E.C.(_50) value of 13.2 ± 0.6 mM. Experiments were performed to investigate in vivo CO(_2) stimulation of Rv1625c(_204-443) transformed into Escherichia coli cells. The results showed that in vivo Rv1625c(_204-443) is stimulated by 10 % (v/v) CO(_2) in air. CO(_2) stimulation of Rv1625c(_204-443) was further investigated by identifying the conditions required for CO(_2) to bind to Rv1625c(_204-443). Numerous spectroscopic and biochemical techniques were used to identify conditions required for CO(_2) binding such as circular dichroism spectroscopy, Fourier transform infrared spectroscopy, HCO(_3) dependent luminescence probe spectroscopy, CO(_2) stimulation assays on Rv1625c(_204-443) mutant proteins, and CO(_2) binding assays. The results showed that metal is not required for CO(_2) binding. CO(_2) binds solely to the apoprotein. As CO(_2) does not require any cefaclors or substrate to bind to Rv1625c(_204-443), the elucidation of the CO(_2) binding site on Rv1625c(_204-443) was attempted. Numerous binding sites were investigated, such as amino acids in the active site with a primary or secondary amine side group (forming a potential carbamate with CO(_2)). Double and triple amino acid CO(_2) binding sites were also investigated by identifying the amino acids that conespond to the HCO(_3) binding site from mammalian soluble adenylyl cyclase. CO(_2) binding was also investigated by growing Rv1625c crystals, exposing the crystals to CO(_2) and X-ray diffraction studies on the crystals to obtain a diffraction data set. Unfortunately none of these techniques was able to identify the CO(_2_ binding site of Rv1625c.This surprising and novel finding that CO’_2’ binds and stimulates Rv1625c(_204-443) suggests that other Class IIIa adenylyl cyclases, including the heterotrimeric G-protein stimulated mammalian adenylyl cyclases are also stimulated by CO(_2) and further work needs to be done in this area.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||08 Sep 2011 18:26|