Literature summary for 3.4.17.12 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.

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Cloned(Commentary)

Cloned (Comment)	Organism
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

Cloned (Comment)

Organism

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

Protein Variants	Comment	Organism
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
E260Q	mutation has minimal effects on kinetic parameters, but decreased heat stability	Homo sapiens
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
S406A	very similar to the wild-type enzyme with regard to expression and release by phosphatidylinositol-specific phospholipase C	Homo sapiens
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
S406T	very similar to the wild-type enzyme with regard to expression and release by phosphatidylinositol-specific phospholipase C	Homo sapiens

Inhibitors

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

KM Value [mM]

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

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
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

Metals/Ions	Comment	Organism
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
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
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
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
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]

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

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	-	-	-

Posttranslational Modification

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

Source Tissue

Source Tissue	Comment	Organism	Textmining
placenta	-	Homo sapiens	-

Substrates and Products (Substrate)

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

Subunits

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

Temperature Stability [°C]

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

Turnover Number [1/s]

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

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6.5	7.5	-	Homo sapiens