The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.
beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.
Shi
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Departments of Biological Chemistry and Medicinal Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Chen
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Yin
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KC
Na
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Garsky
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Lai
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Li
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Platchek
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Register
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Sardana
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Tang
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MJ
Thiebeau
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Wood
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Shafer
J A
JA
Gardell
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D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D003208:Concanavalin A / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D045683:Furin; D019791:Guanidine / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011489:Protein Denaturation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013381:Subtilisins / Q000494:pharmacology; D013997:Time Factors
D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D011498:Protein Precursors; D011994:Recombinant Proteins; D003208:Concanavalin A; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D013381:Subtilisins; D045683:Furin; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human; C509280:BACE1 protein, human; D019791:Guanidine