Determination of the Degree of Channeling Present between Separate Catalytic Domains in Rat Peptidyl-Glycine Alpha-Amidating Monooxygenase

Determination of the Degree of Channeling Present between Separate Catalytic Domains in Rat Peptidyl-Glycine Alpha-Amidating Monooxygenase

Rat Peptidyl-Gly Alpha-Amidating Monooxygenase, or PAM, is the ortholog of the enzyme in humans solely responsible for amidating bioactive peptides to enable full biological function. The enzyme is composed of two catalytic domains held together by a flexible linker. The first domain is the copper-containing peptidylglycine α-hydroxylating monooxygenase, or PHM, which α-hydroxylates the peptidyl-glycine substrates. The second domain is the zinc-containing peptidyl-α- hydroxyglycine α-amidating lyase, or PAL, which produces glyoxylate and the amidated peptide final product. In part due to the flexible linker between the domains, the mechanism of substrate transfer between PHM and PAL is poorly characterized. Channeling, or direct substrate transfer, is a method used by enzymes to prevent release of intermediates into solution when the intermediate is easily degraded or otherwise unstable. We use the ratio between the oxygen consumed by the PHM domain and the glyoxylate produced by the PAL domain to determine the percentage at which successful substrate transfer occurs, which indicates whether direct transfer via channeling or release of intermediates into solution occurs. Using substrates of different amino acid sequence, oxygen consumption was measured via Oxygraph to determine the rate of oxygen consumption by the PHM domain, which was compared to the glyoxylate production at the same timepoints by the PAL domain. These data will be used to draw conclusions about primary intermediate transfer mechanism.