The role of α-amino group of the N-terminal serine of β subunit for enzyme catalysis and autoproteolytic activation of glutaryl 7-aminocephalosporanic acid acylase

Sik Lee Young Sik Lee, Wook Kim Hyung Wook Kim, Sung Soo Park

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 catalyzes the cleavage of the amide bond in the GL-7-ACA side chain to produce glutaric acid and 7-aminocephalosporanic acid (7-ACA). The active enzyme is an (αβ)2 heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. In this study, we prepared and characterized a chemically modified enzyme, and also examined an effect of the modification on enzyme catalysis and autocatalytic processing of the enzyme precursor. We found that treatment of the enzyme with cyanate ion led to a significant loss of the enzyme activity. Structural and functional analyses of the modified enzyme showed that carbamylation of the free α-amino group of the N-terminal Ser-199 of the β subunit resulted in the loss of the enzyme activity. The pH dependence of the kinetic parameters indicates that a single ionizing group is involved in enzyme catalysis with pKa = 6.0, which could be attributed to the α-amino group of the N-terminal Ser-199. The carbamylation also inhibited the secondary processing of the enzyme precursor, suggesting a possible role of the α-amino group for the reaction. Mutagenesis of the invariant N-terminal residue Ser-199 confirmed the key function of its side chain hydroxyl group in both enzyme catalysis and autoproteolytic activation. Partial activity and correct processing of a mutant S199T were in agreement with the general mechanism of N-terminal nucleophile hydrolases. Our results indicate that GL-7-ACA acylase utilizes as a nucleophile Ser-199 in both enzyme activity and autocatalytic processing and most importantly its own α-amino group of the Ser-199 as a general base catalyst for the activation of the hydroxyl group both in enzyme catalysis and in the secondary cleavage of the enzyme precursor. All of the data also imply that GL-7-ACA acylase is a member of a novel class of N-terminal nucleophile hydrolases that have a single catalytic center for enzyme catalysis.

Original languageEnglish
Pages (from-to)39200-39206
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number50
DOIs
Publication statusPublished - 2000 Dec 15

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amidase
Catalysis
Serine
Chemical activation
Enzymes
Enzyme Precursors
Enzyme activity
Processing
Hydroxyl Radical
Cyanates
glutaryl-7-aminocephalosporanic acid
Mutagenesis
Nucleophiles
Kinetic parameters
Amides

ASJC Scopus subject areas

  • Biochemistry

Cite this

The role of α-amino group of the N-terminal serine of β subunit for enzyme catalysis and autoproteolytic activation of glutaryl 7-aminocephalosporanic acid acylase. / Young Sik Lee, Sik Lee; Hyung Wook Kim, Wook Kim; Park, Sung Soo.

In: Journal of Biological Chemistry, Vol. 275, No. 50, 15.12.2000, p. 39200-39206.

Research output: Contribution to journalArticle

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title = "The role of α-amino group of the N-terminal serine of β subunit for enzyme catalysis and autoproteolytic activation of glutaryl 7-aminocephalosporanic acid acylase",
abstract = "Glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 catalyzes the cleavage of the amide bond in the GL-7-ACA side chain to produce glutaric acid and 7-aminocephalosporanic acid (7-ACA). The active enzyme is an (αβ)2 heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. In this study, we prepared and characterized a chemically modified enzyme, and also examined an effect of the modification on enzyme catalysis and autocatalytic processing of the enzyme precursor. We found that treatment of the enzyme with cyanate ion led to a significant loss of the enzyme activity. Structural and functional analyses of the modified enzyme showed that carbamylation of the free α-amino group of the N-terminal Ser-199 of the β subunit resulted in the loss of the enzyme activity. The pH dependence of the kinetic parameters indicates that a single ionizing group is involved in enzyme catalysis with pKa = 6.0, which could be attributed to the α-amino group of the N-terminal Ser-199. The carbamylation also inhibited the secondary processing of the enzyme precursor, suggesting a possible role of the α-amino group for the reaction. Mutagenesis of the invariant N-terminal residue Ser-199 confirmed the key function of its side chain hydroxyl group in both enzyme catalysis and autoproteolytic activation. Partial activity and correct processing of a mutant S199T were in agreement with the general mechanism of N-terminal nucleophile hydrolases. Our results indicate that GL-7-ACA acylase utilizes as a nucleophile Ser-199 in both enzyme activity and autocatalytic processing and most importantly its own α-amino group of the Ser-199 as a general base catalyst for the activation of the hydroxyl group both in enzyme catalysis and in the secondary cleavage of the enzyme precursor. All of the data also imply that GL-7-ACA acylase is a member of a novel class of N-terminal nucleophile hydrolases that have a single catalytic center for enzyme catalysis.",
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T1 - The role of α-amino group of the N-terminal serine of β subunit for enzyme catalysis and autoproteolytic activation of glutaryl 7-aminocephalosporanic acid acylase

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AU - Hyung Wook Kim, Wook Kim

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N2 - Glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 catalyzes the cleavage of the amide bond in the GL-7-ACA side chain to produce glutaric acid and 7-aminocephalosporanic acid (7-ACA). The active enzyme is an (αβ)2 heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. In this study, we prepared and characterized a chemically modified enzyme, and also examined an effect of the modification on enzyme catalysis and autocatalytic processing of the enzyme precursor. We found that treatment of the enzyme with cyanate ion led to a significant loss of the enzyme activity. Structural and functional analyses of the modified enzyme showed that carbamylation of the free α-amino group of the N-terminal Ser-199 of the β subunit resulted in the loss of the enzyme activity. The pH dependence of the kinetic parameters indicates that a single ionizing group is involved in enzyme catalysis with pKa = 6.0, which could be attributed to the α-amino group of the N-terminal Ser-199. The carbamylation also inhibited the secondary processing of the enzyme precursor, suggesting a possible role of the α-amino group for the reaction. Mutagenesis of the invariant N-terminal residue Ser-199 confirmed the key function of its side chain hydroxyl group in both enzyme catalysis and autoproteolytic activation. Partial activity and correct processing of a mutant S199T were in agreement with the general mechanism of N-terminal nucleophile hydrolases. Our results indicate that GL-7-ACA acylase utilizes as a nucleophile Ser-199 in both enzyme activity and autocatalytic processing and most importantly its own α-amino group of the Ser-199 as a general base catalyst for the activation of the hydroxyl group both in enzyme catalysis and in the secondary cleavage of the enzyme precursor. All of the data also imply that GL-7-ACA acylase is a member of a novel class of N-terminal nucleophile hydrolases that have a single catalytic center for enzyme catalysis.

AB - Glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 catalyzes the cleavage of the amide bond in the GL-7-ACA side chain to produce glutaric acid and 7-aminocephalosporanic acid (7-ACA). The active enzyme is an (αβ)2 heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. In this study, we prepared and characterized a chemically modified enzyme, and also examined an effect of the modification on enzyme catalysis and autocatalytic processing of the enzyme precursor. We found that treatment of the enzyme with cyanate ion led to a significant loss of the enzyme activity. Structural and functional analyses of the modified enzyme showed that carbamylation of the free α-amino group of the N-terminal Ser-199 of the β subunit resulted in the loss of the enzyme activity. The pH dependence of the kinetic parameters indicates that a single ionizing group is involved in enzyme catalysis with pKa = 6.0, which could be attributed to the α-amino group of the N-terminal Ser-199. The carbamylation also inhibited the secondary processing of the enzyme precursor, suggesting a possible role of the α-amino group for the reaction. Mutagenesis of the invariant N-terminal residue Ser-199 confirmed the key function of its side chain hydroxyl group in both enzyme catalysis and autoproteolytic activation. Partial activity and correct processing of a mutant S199T were in agreement with the general mechanism of N-terminal nucleophile hydrolases. Our results indicate that GL-7-ACA acylase utilizes as a nucleophile Ser-199 in both enzyme activity and autocatalytic processing and most importantly its own α-amino group of the Ser-199 as a general base catalyst for the activation of the hydroxyl group both in enzyme catalysis and in the secondary cleavage of the enzyme precursor. All of the data also imply that GL-7-ACA acylase is a member of a novel class of N-terminal nucleophile hydrolases that have a single catalytic center for enzyme catalysis.

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