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Literature summary extracted from

  • Tan, F.; Balsitis, S.; Black, J.K.; Blochl, A.; Mao, J.F.; Becker, R.P.; Schacht, D.; Skidgel, R.A.
    Effect of mutation of two critical glutamic acid residues on the activity and stability of human carboxypeptidase M and characterization of its signal for glycosylphosphatidylinositol anchoring (2003), Biochem. J., 370, 567-578.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.4.17.12 expression of wild-type and mutant enzymes in COS-7 cells or HEK-293 cells. The wild-type and S406A and S406T mutants are expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, the S406P mutant is notz and is retained in a perinuclear location. Expression in baculovirus infected cells in a glycophosphatidyl-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidyl anchoring is secreted at high levels into the medium. Both forms have lower molecular masses than native placental enzyme indicating a minimal glycosylation Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
3.4.17.12 E260A mutation reduces the ratio of turnover-numver to Km-value by 104fold and further decreases stability.Addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wild-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297% Homo sapiens
3.4.17.12 E260Q mutation has minimal effects on kinetic parameters, but decreased heat stability Homo sapiens
3.4.17.12 E264Q mutation results in a 10000fold decrease in activity, but the enzyme still binds to p-aminobenzoylarginine-Sepharose and is resistant to trypsin treatment, indicating that the protein is folded properly Homo sapiens
3.4.17.12 S406A very similar to the wild-type enzyme with regard to expression and release by phosphatidylinositol-specific phospholipase C Homo sapiens
3.4.17.12 S406P the wild-type and S406A and S406T mutants are expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, the S406P mutant is not and is retained in a perinuclear location Homo sapiens
3.4.17.12 S406T very similar to the wild-type enzyme with regard to expression and release by phosphatidylinositol-specific phospholipase C Homo sapiens

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.4.17.12 1,10-phenanthroline 1 mM, 96% inhibition Homo sapiens
3.4.17.12 Guanidinoethyl mercaptosuccinic acid 1 mM, 97% inhibition Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.4.17.12 0.059
-
 Dansyl-Ala-Arg wild-type enzyme lacking the C-terminal hydrophobic membrane anchor signal Homo sapiens
3.4.17.12 0.102
-
 Dansyl-Ala-Arg mutant enzyme E260Q Homo sapiens
3.4.17.12 0.28
-
 Dansyl-Ala-Arg mutant enzyme E260A Homo sapiens

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
3.4.17.12 plasma membrane expression of wild-type and mutant enzymes in COS-7 cells or HEK-293 cells.The wild-type and S406A and S406T mutants are expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, the S406P mutant is not and is retained in a perinuclear location Homo sapiens 5886
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.4.17.12 KCl addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297% Homo sapiens
3.4.17.12 KNO3 addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297% Homo sapiens
3.4.17.12 Na2SO4 addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297% Homo sapiens
3.4.17.12 NaCl addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297% Homo sapiens
3.4.17.12 NaNO3 addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297% Homo sapiens

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.4.17.12 50000
-
 x * 50000, SDS-PAGE Homo sapiens

Organism

EC Number Organism UniProt Comment Textmining
3.4.17.12 Homo sapiens
-
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
3.4.17.12 glycoprotein the wild-type enzyme lacking the C-terminal hydrophobic membrane anchor signal contains only 5.4% carbohydrate Homo sapiens
3.4.17.12 glycoprotein a truncated form, lacking the putative signal sequence for glycosylphosphatidyl anchoring is secreted at high levels into the medium Homo sapiens

Source Tissue

EC Number Source Tissue Comment Organism Textmining
3.4.17.12 placenta
-
 Homo sapiens
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.4.17.12 Dansyl-Ala-Arg + H2O
-
 Homo sapiens Dansyl-Ala + Arg
-
 ?

Subunits

EC Number Subunits Comment Organism
3.4.17.12 ? x * 50000, SDS-PAGE Homo sapiens

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.4.17.12 37
-
 wild-type enzyme and mutant enzymes E260Q and E260A are stable Homo sapiens
3.4.17.12 45
-
 15 min, 50% inactivation of mutant enzyme E260A Homo sapiens
3.4.17.12 49
-
 15 min, 15 min, 50% inactivation of mutant enzyme E260Q Homo sapiens
3.4.17.12 50
-
 15 min, complete inactivation of mutant enzyme E260A Homo sapiens
3.4.17.12 53
-
 15 min, wild-type enzyme retains 50% of its activity Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.4.17.12 0.233
-
 Dansyl-Ala-Arg mutant enzyme E260A Homo sapiens
3.4.17.12 5.12
-
 Dansyl-Ala-Arg wild-type enzyme lacking the C-terminal hydrophobic membrane anchor signal Homo sapiens
3.4.17.12 6.02
-
 Dansyl-Ala-Arg mutant enzyme E260Q Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.4.17.12 6.5 7.5
-
 Homo sapiens