Both amidated and nonamidated forms of glucagon-like peptide I are synthesized in the rat intestine and the pancreas.

Biologically active peptides are initially synthesized in the form of protein precursors, and the peptides are liberated by post-translational processing from the precursors in a tissue-specific manner. Mammalian proglucagon, which is synthesized in the neuroendocrine L-cells of the intestine and the alpha-cells of the pancreas, contains within its structure the sequences of glucagon and two glucagon-like peptides (GLP-I and GLP-II) flanked at their amino and carboxyl termini by dibasic residues. Tissue-specific processing liberates different peptides in the intestine compared with the pancreas. One of these intestinal peptides, glucagon-like peptide I(7-37) (GLP-I(7-37], is one of the most potent insulin secretagogues studied to date. It contains within its carboxyl-terminal domain an arginine residue that, because of an adjacent glycine residue, may alternatively be used during post-translational processing as a site for amidation. Using a chromatographic system and radioimmunoassays that discriminate among the closely related GLP-I peptides, we find that the processing of proglucagon in the rat intestine and to a lesser extent in the rat pancreas results in the formation of at least three GLP-I peptides, of 37, 31, and 30 residues. The 30-residue peptide is in the form of an alpha-carboxyl-terminal arginine amide, a modification that is not usually found in proteins. Remarkably, the relative potencies for the stimulation of insulin secretion from the perfused rat pancreas of the nonamidated (GLP-I(7-37] and the amidated (GLP-I(7-36) amide) peptides are the same (Weir, G. C., Mojsov, S., Hendrik, G. K., and Habener, J. F. (1989) Diabetes 38, 338-342; Suzuki, S., Kawai, K., Okashir, S., Mukal, H., and Yamashita, K. (1989) Endocrinology 125, 3109-3114).

Electron spin-echo studies of the composition of the paramagnetic intermediate formed during the deamination of propanolamine by ethanolamine ammonia-lyase, and AdoCbl-dependent enzyme.

During the deamination of S-2-aminopropanol by the AdoCbl-dependent ethanolamine ammonia-lyase of Clostridia sp., a catalytic intermediate accumulates whose active site contains two paramagnetic species: cob(II)alamin and a free radical derived from the substrate molecule. Spin-echo spectroscopy has revealed that the unpaired electron on the substrate-derived radical is delocalized over a nitrogen atom that from its quadrupole splittings is probably a component of a secondary amide group. Experiments with 15N- and deuterium-labeled propanolamine gave no evidence of an interaction between this unpaired electron and the nitrogen originally attached to the substrate molecule. These results strongly suggest that the substrate-derived radical in this intermediate has already lost its nitrogen, and that this radical is stabilized by delocalization of the unpaired electron onto a nitrogen most likely situated in one of the peptide bonds of the enzyme backbone.

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence that the hydrogen transfer mechanism involves a second intermediate hydrogen carrier in addition to the cofactor.

Ethanolamine ammonia-lyase catalyzes the adenosylcobalamin (AdoCbl)-dependent conversion of ethanolamine to acetaldehyde and ammonia. During this reaction, a hydrogen atom migrates from the carbinol carbon of ethanolamine to the methyl carbon of acetaldehyde. Previous studies have shown that this migrating hydrogen equilibrates with the hydrogens on the 5'-(cobalt-linked) carbon of the cofactor. On the basis of those studies, a two-step mechanism for hydrogen transfer has been postulated in which the migrating hydrogen is first transferred from the substrate to the cofactor, then in a subsequent step is returned from the cofactor to the product. We now show that this migrating hydrogen is transferred not only to the cofactor, but also to a second acceptor at the active site. Hydrogens on this acceptor do not exchange with water during the course of the reaction, but are released to water when the enzyme is denatured. The catalytic significance of this second hydrogen acceptor was demonstrated by the findings that the transfer of hydrogen to this acceptor required both AdoCbl and active enzyme and that hydrogen at the second acceptor site could be washed out by unlabeled ethanolamine. On the basis of these results, we propose an expanded hydrogen transfer mechanism in which AdoCbl and the second acceptor site serve as alternative intermediate hydrogen carriers during the course of ethanolamine deamination.

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence for a carboxyl group at the active site.

Ethanolamine ammonia-lyase is an adenosylcobalamin-dependent enzyme that catalyzes the rearrangement of ethanolamine and other vicinal amino alcohols to oxo-compounds and ammonia. Treatment of this enzyme with the sulfhydryl group-blocking reagent methyl methanethiosulfonate produces a species with diminished catalytic activity. When methyl methanethiosulfonate -treated ethanolamine ammonia-lyase was incubated with a carboxyl-blocking reagent consisting of glycine ethyl ester plus a water-soluble carbodiimide, the enzyme lost more than 80% of its residual activity, while at the same time glycine ethyl ester was incorporated into it at a stoichiometry of 6 mol/mol of enzyme. Both the loss of activity and the incorporation of glycine ethyl ester were prevented if ethanolamine was included in the glycine ethyl ester-containing incubation mixture. These results suggest that an active site carboxyl group plays a role in the mechanism of catalysis by ethanolamine ammonia-lyase, and that this carboxyl group is amidated when the enzyme is incubated with glycine ethyl ester plus carbodiimide.