Enzymic activities of two-chain pepsinogen, two-chain pepsin, and the amino-terminal lobe of pepsinogen.
In order to study the relationships of aspartic proteases, we have modified pepsin, a single-chain eukaryotic enzyme, to a two-chain heterodimer, which resembles aspartic proteases from retrovirus, including human immunodeficiency virus. Two fragments of pepsinogen, residues 1P-172 and 173-326, were expressed separately in Escherichia coli. Mixtures of chains were refolded from urea solutions to generate an active two-chain pepsinogen, which was converted to two-chain pepsin in acid solutions. The intramolecular and bimolecular activation constants (k1 and k2) of two-chain pepsinogen are about 1.5-fold and one-sixth, respectively, of those for pepsinogen. Structural evidence suggests that the faster k1 of two-chain pepsinogen is due to decreased interaction of the propeptide with the pepsin moiety, implying that the rate-limiting step in the intramolecular activation of pepsinogen is the "conformational dissociation" of its propeptide. Two-chain pepsin has the same Km but only one-sixth of the kcat of pepsin. Both pepsinogen chains are capable of independent refolding. The refolding of the NH2-terminal chain, which contains the propeptide and the NH2-terminal lobe, generated a small amount of proteolytic activity which is likely derived from the homodimer of the NH2-terminal lobe. It has been postulated that mammalian aspartic proteases, which contain two structurally homologous lobes, are derived in evolution from a homodimer enzyme by gene duplication and fusion (Tang, J., James, M. N. G., Hsu, I.-N., Jenkins, J. A., and Blundell, T. L. (1978) Nature 271, 618-621). The observation of the homodimer activity of the NH2-terminal lobe of pepsinogen suggests that the interface of the lobes is conservative in evolution.
Lin
X L
XL
Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Lin
Y Z
YZ
Koelsch
G
G
Gustchina
A
A
Wlodawer
A
A
Tang
J
J
D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003196:Computer Graphics; D005822:Genetic Vectors; D016333:HIV Protease / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010434:Pepsin A / Q000737:chemistry / Q000378:metabolism*; D010435:Pepsinogens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010435:Pepsinogens; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016333:HIV Protease; D010434:Pepsin A