THE IDENTIFICATION OF NOVEL INORGANIC CARBON SENSITIVE ENZYMES

Abstract

Adenylyl cyclase catalyses the formation of the second messenger adenosine-3’, 5’-monophosphate from adenosine triphosphate, and is involved in a number of diverse signalling pathways in eukaryotes and prokaryotes. Adenylyl cyclases are diverse in their structure and biochemistry, and have been grouped into six distinct Classes (I–VI). The Class III cyclase homology domain comprises the majority of prokaryotic and eukaryotic adenylyl cyclases, and has been further divided into 4 sub-Classes (a-d) based on active site polymorphisms. A number of Class IIIb adenylyl cyclases display elevated catalytic activity in the presence of inorganic carbon. Whether a response to inorganic carbon can be observed in enzymes which do not possess a Class IIIb cyclase homology domain remains to be established.
Experiments were performed to investigate the response to inorganic carbon of a Class IIIa cyclase homology domain; mammalian transmembrane adenylyl cyclase. In vivo experiments demonstrated that the activity of mammalian transmembrane adenylyl cyclase was potentially regulated by inorganic carbon, and that this had a downstream effect on the cAMP response element binding protein. In vitro experiments performed on a transmembrane adenylyl cyclase demonstrated that the increase in activity in the presence of inorganic carbon occurred through an increase in kcat and increased metal affinity.
Experiments were performed to test the response to inorganic carbon of several enzymes that share a structurally similar active site with the adenylyl cyclases; the polymerase I family of prokaryotic DNA polymerases, the polymerase β family of DNA polymerases, and the guanylyl cyclases. Initial in vitro experiments performed on T7 RNA polymerase demonstrated a response to inorganic carbon, however, it was discovered that this was likely due to a non-specific effect of pH. It was shown that inorganic carbon increased assay pH over time, and this warranted a re-design of the in vitro assay used to test the response of an enzyme to inorganic carbon. This new in vitro assay methodology was used to re-test T7 RNA polymerase, as well as test DNA polymerase β and several guanylyl cyclase, and demonstrated that these enzymes were non-responsive to inorganic carbon.
Using this newly devised in vitro assay, experiments were performed to re-test the response of mammalian transmembrane adenylyl cyclase to inorganic carbon, and demonstrated that this enzyme was unlikely to be regulated by inorganic carbon. Furthermore, this new in vitro assay was used to re-test the response of several Class IIIb cyclase homology domains to inorganic carbon. Results demonstrated that mammalian soluble adenylyl cyclase was responsive to inorganic carbon, however, results provided evidence to suggest that two prokaryotic Class IIIb cyclase homology domains (CyaB1 from Anabaena PCC 7120 and CyaC from Spirulina platensis) were possibly non-responsive to inorganic carbon.