DB code: S00437

RLCP classification	1.13.30185.55 : Hydrolysis
CATH domain	3.60.60.10 : Penicillin V Acylase; Chain A	Catalytic domain
E.C.	3.5.1.11
CSA
M-CSA
MACiE	M0241

CATH domain	Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	MEROPS	Pfam
P12256	Penicillin acylase	EC 3.5.1.11 Penicillin V amidase PVA	C59.001 (Cysteine)	PF02275 (CBAH) [Graphical View]

KEGG enzyme name
penicillin amidase penicillin acylase benzylpenicillin acylase novozym 217 semacylase alpha-acylamino-beta-lactam acylhydrolase ampicillin acylase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P12256	PAC_BACSH	Penicillin + H(2)O = a carboxylate + 6- aminopenicillanate.	Homotetramer.

KEGG Pathways
Map code	Pathways	E.C.
MAP00311	Penicillin and cephalosporin biosynthesis

Compound table
	Substrates		Products		Intermediates
KEGG-id	C00395	C00001	C00060	C02954
E.C.
Compound	Penicillin	H2O	Carboxylate	6-Aminopenicillanate
Type	carboxyl group,peptide/protein,sulfide group	H2O	carboxyl group	amide group,amine group,carboxyl group,sulfide group
ChEBI		15377 15377		16705 57869 16705 57869
PubChem		22247451 962 22247451 962		11082 7057887 11082 7057887

2pvaA	Unbound		Unbound	Unbound
2pvaB	Unbound		Unbound	Unbound
2pvaC	Unbound		Unbound	Unbound
2pvaD	Unbound		Unbound	Unbound
3pvaA	Unbound		Unbound	Unbound
3pvaB	Unbound		Unbound	Unbound
3pvaC	Unbound		Unbound	Unbound
3pvaD	Unbound		Unbound	Unbound
3pvaE	Unbound		Unbound	Unbound
3pvaF	Unbound		Unbound	Unbound
3pvaG	Unbound		Unbound	Unbound
3pvaH	Unbound		Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
literature [1] & [4]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

2pvaA	;ASP 20;ASN 175;ARG 228		OCS 1	TYR 82	OCS,Cysteinesulfonic acid
2pvaB	;ASP 20;ASN 175;ARG 228		OCS 1	TYR 82	OCS,Cysteinesulfonic acid
2pvaC	;ASP 20;ASN 175;ARG 228		OCS 1	TYR 82	OCS,Cysteinesulfonic acid
2pvaD	;ASP 20;ASN 175;ARG 228		OCS 1	TYR 82	OCS,Cysteinesulfonic acid
3pvaA	CYS 1;ASP 20;ASN 175;ARG 228			TYR 82
3pvaB	CYS 1;ASP 20;ASN 175;ARG 228			TYR 82
3pvaC	CYS 1;ASP 20;ASN 175;ARG 228			TYR 82
3pvaD	CYS 1;ASP 20;ASN 175;ARG 228			TYR 82
3pvaE	CYS 1;ASP 20;ASN 175;ARG 228			TYR 82
3pvaF	CYS 1;ASP 20;ASN 175;ARG 228			TYR 82
3pvaG	CYS 1;ASP 20;ASN 175;ARG 228			TYR 82
3pvaH	CYS 1;ASP 20;ASN 175;ARG 228			TYR 82

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[1]	Fig.3	5
[3]	p.239
[4]	p.414
[6]	Fig.2	3
[7]	p.146-147

References
[1]
Resource
Comments	Penicillin G acylase (homologous enzyme)
Medline ID
PubMed ID	7816145
Journal	Nature
Year	1995
Volume	373
Pages	264-8
Authors	Duggleby HJ, Tolley SP, Hill CP, Dodson EJ, Dodson G, Moody PC
Title	Penicillin acylase has a single-amino-acid catalytic centre.
Related PDB
Related UniProtKB
[2]
Resource
Comments	Penicillin G acylase (homologous enzyme)
Medline ID
PubMed ID	7477383
Journal	Nature
Year	1995
Volume	378
Pages	416-9
Authors	Brannigan JA, Dodson G, Duggleby HJ, Moody PC, Smith JL, Tomchick DR, Murzin AG
Title	A protein catalytic framework with an N-terminal nucleophile is capable of self-activation.
Related PDB
Related UniProtKB
[3]
Resource
Comments	Penicillin G acylase (homologous enzyme)
Medline ID
PubMed ID	9931321
Journal	Biochem J
Year	1999
Volume	338
Pages	235-9
Authors	Morillas M, Goble ML, Virden R
Title	The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre.
Related PDB
Related UniProtKB
[4]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	10331865
Journal	Nat Struct Biol
Year	1999
Volume	6
Pages	414-416
Authors	Suresh CG, Pundle AV, SivaRaman H, Rao KN, Brannigan JA, McVey CE, Verma CS, Dauter Z, Dodson EJ, Dodson GG
Title	Penicillin V acylase crystal structure reveals new Ntn-hydrolase family members.
Related PDB	2pva 3pva
Related UniProtKB
[5]
Resource
Comments	Penicillin G acylase (homologous enzyme)
Medline ID
PubMed ID	10993730
Journal	J Mol Biol
Year	2000
Volume	302
Pages	887-98
Authors	Hewitt L, Kasche V, Lummer K, Lewis RJ, Murshudov GN, Verma CS, Dodson GG, Wilson KS
Title	Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft.
Related PDB
Related UniProtKB
[6]
Resource
Comments	X-ray crystallography, catalysis; Penicillin G acylase (homologous enzyme)
Medline ID
PubMed ID	11239085
Journal	Protein Eng
Year	2000
Volume	13
Pages	857-863
Authors	Alkema WB, Hensgens CM, Kroezinga EH, de Vries E, Floris R, van der Laan JM, Dijkstra BW, Janssen DB
Title	Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies.
Related PDB
Related UniProtKB
[7]
Resource
Comments	X-ray crystallography (1.3 Angstroms), catalysis; Penicillin G acylase (homologous enzyme)
Medline ID
PubMed ID	11601852
Journal	J Mol Biol
Year	2001
Volume	313
Pages	139-150
Authors	McVey CE, Walsh MA, Dodson GG, Wilson KS, Brannigan JA
Title	Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism.
Related PDB
Related UniProtKB

Comments
This enzyme belongs to N-terminal nucleophile (Ntn) hydrolase family, of which sidechain of the N-terminal residue acts as nucleophile, assisted by its own alpha-amino group. The paper [1] proposed the catalytic mechanism of the homologous enzyme, penicillin G acylase, whose nucleophilic residue is serine instead of cysteine. (1) The alpha-amino group enhances the nucleophilicity of the sidechain of the N-terminal residue, through the bridging water between them. (2) The sidechain of Cys1 makes a nucleophilic attack on the acyl carbon of penicillin, forming an oxyanion tetrahedral, stabilized by the oxyanion hole composed of mainchain amides and sidechain of Asn175. Here, the alpha-amino group protonates the leaving amine group through a water. (3) This tetrahedral intermediate will then collapse to form an acyl enzyme and release the free 6-aminopenicillanic acid. (4) The acyl enzyme will be attacked by water to form a second tetrahedral intermediate, stabilized by the same oxyanion hole, which can in turn collapse to release the free phenylacetic acid. In contrast, the literature [7] suggested that the catalytic mechanism proceeds via direct nucleophilic attack of the nucleophilic residue on the scissile amide and not as via the bridging water molecule acting as a "virtual" base. According to the paper [3], the residue corresponding to Arg228 in its homologue, penicillin G acylase, seems to be important for catalysis, orienting the N-terminal catalytic residue and contributing to a decrease in the pKa of alpha-amino group. The literature on this enzyme [4] reported that the oxyanion hole consists of the sidechain of Asn175 and NH of Tyr82, whilst Arg228, Asp20 and Asn175 are critical for positioning the lone pair of the unprotonated N-terminal alpha-amino group. This might lead to the decrease in the pKa of the alpha-amino group.

Comments

This enzyme belongs to N-terminal nucleophile (Ntn) hydrolase family, of which sidechain of the N-terminal residue acts as nucleophile, assisted by its own alpha-amino group.
The paper [1] proposed the catalytic mechanism of the homologous enzyme, penicillin G acylase, whose nucleophilic residue is serine instead of cysteine.
(1) The alpha-amino group enhances the nucleophilicity of the sidechain of the N-terminal residue, through the bridging water between them.
(2) The sidechain of Cys1 makes a nucleophilic attack on the acyl carbon of penicillin, forming an oxyanion tetrahedral, stabilized by the oxyanion hole composed of mainchain amides and sidechain of Asn175. Here, the alpha-amino group protonates the leaving amine group through a water.
(3) This tetrahedral intermediate will then collapse to form an acyl enzyme and release the free 6-aminopenicillanic acid.
(4) The acyl enzyme will be attacked by water to form a second tetrahedral intermediate, stabilized by the same oxyanion hole, which can in turn collapse to release the free phenylacetic acid.
In contrast, the literature [7] suggested that the catalytic mechanism proceeds via direct nucleophilic attack of the nucleophilic residue on the scissile amide and not as via the bridging water molecule acting as a "virtual" base.
According to the paper [3], the residue corresponding to Arg228 in its homologue, penicillin G acylase, seems to be important for catalysis, orienting the N-terminal catalytic residue and contributing to a decrease in the pKa of alpha-amino group.
The literature on this enzyme [4] reported that the oxyanion hole consists of the sidechain of Asn175 and NH of Tyr82, whilst Arg228, Asp20 and Asn175 are critical for positioning the lone pair of the unprotonated N-terminal alpha-amino group. This might lead to the decrease in the pKa of the alpha-amino group.

Created	Updated
2002-09-04	2009-02-26