Acidic pH as a physiological regulator of human cathepsin L activity.
Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate-independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in alpha-helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ.mol-1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in-vivo regulation and turnover of cathepsin L inside lysosomes.
Turk
B
B
Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia. boris.turk@ijs.si
Dolenc
I
I
Lenarcic
B
B
Krizaj
I
I
Turk
V
V
Bieth
J G
JG
Björk
I
I
D000595:Amino Acid Sequence; D002402:Cathepsin D / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry*; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D010450:Endopeptidases; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012997:Solvents / Q000494:pharmacology; D013696:Temperature
D012997:Solvents; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; D002402:Cathepsin D; D004121:Dimethyl Sulfoxide