Arg(1098) is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity.
Angiotensin (Ang) I-converting enzyme (ACE) is a Zn(2+) metalloprotease with two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl(-), but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl(-) dependence. Cl(-) dependence is also lost when the equivalent Arg in the N-domain, Arg(500), is substituted with Gln. The Arg(1098) to Lys substitution reduced Cl(-) binding affinity by approximately 100-fold. In the absence of Cl(-), substrate binding affinity (1/K(m)) of and catalytic efficiency (k(cat)/K(m)) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg(1098) to Gln substitution, and are similar (<2-fold difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl(-)]. The Arg(1098) to Gln substitution also eliminates Cl(-) dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wild-type C-domain in the absence of Cl(-). These findings indicate that: 1) Arg(1098) is a critical residue of the C-domain Cl(-)-binding site and 2) a basic side chain is necessary for Cl(-) dependence. For tetrapeptide substrates, the inability of R1098Q to recreate the high affinity state generated by the Cl(-)-C-domain interaction suggests that substrate interactions with the enzyme-bound Cl(-) are much more important for the hydrolysis of short substrates than for Ang I. Since Cl(-) concentrations are saturating under physiological conditions and Arg(1098) is not critical for Ang I hydrolysis, we speculate that the evolutionary pressure for the maintenance of the Cl(-)-binding site is its ability to allow cleavage of short cognate peptide substrates at high catalytic efficiencies.
Liu
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Enzyme Research Unit, Victor Chang Cardiac Research Institute, Sydney, New South Wales 2010, Australia.
Fernandez
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Wouters
M A
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Heyberger
S
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Husain
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D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D019556:COS Cells; D002384:Catalysis; D020134:Catalytic Domain; D002713:Chlorine / Q000737:chemistry* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D014162:Transfection
D010455:Peptides; D011994:Recombinant Proteins; D005973:Glutamine; D012965:Sodium Chloride; D002713:Chlorine; D001120:Arginine; D007703:Peptidyl-Dipeptidase A