Comparative studies of active site-ligand interactions among various recombinant constructs of human beta-amyloid precursor protein cleaving enzyme.
Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.
Kopcho
Lisa M
LM
Department of Chemical Enzymology, Hopewell, NJ, USA.
Ma
Jianhong
J
Marcinkeviciene
Jovita
J
Lai
Zhihong
Z
Witmer
Mark R
MR
Cheng
Janet
J
Yanchunas
Joseph
J
Tredup
Jeffrey
J
Corbett
Martin
M
Calambur
Deepa
D
Wittekind
Michael
M
Paruchuri
Manjula
M
Kothari
Dharti
D
Lee
Grace
G
Ganguly
Subinay
S
Ramamurthy
Vidhyashankar
V
Morin
Paul E
PE
Camac
Daniel M
DM
King
Robert W
RW
Lasut
Amy L
AL
Ross
O Harold
OH
Hillman
Milton C
MC
Fish
Barbara
B
Shen
Keqiang
K
Dowling
Randine L
RL
Kim
Young B
YB
Graciani
Nilsa R
NR
Collins
Dale
D
Combs
Andrew P
AP
George
Henry
H
Thompson
Lorin A
LA
Copeland
Robert A
RA
D000544:Alzheimer Disease / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D010450:Endopeptidases; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D008027:Light; D008051:Lipid Bilayers / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D012542:Scattering, Radiation; D013997:Time Factors
D008024:Ligands; D008051:Lipid Bilayers; D010455:Peptides; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human