DB code: S00440

RLCP classification	3.1244.220200.43 : Transfer
CATH domain	3.75.10.10 : L-arginine/glycine Amidinotransferase; Chain A	Catalytic domain
E.C.	2.1.4.1
CSA	1jdw
M-CSA	1jdw
MACiE	M0018

CATH domain	Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	RefSeq	Pfam
P50440	Glycine amidinotransferase, mitochondrial	EC 2.1.4.1 L-arginine:glycine amidinotransferase Transamidinase AT	NP_001473.1 (Protein) NM_001482.2 (DNA/RNA sequence)	PF02274 (Amidinotransf) [Graphical View]

KEGG enzyme name
glycine amidinotransferase arginine-glycine amidinotransferase arginine-glycine transamidinase glycine transamidinase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P50440	GATM_HUMAN	L-arginine + glycine = L-ornithine + guanidinoacetate.	Homodimer. There is an equilibrium between the monomeric and dimeric forms, shifted towards the side of the monomer.	Mitochondrion inner membrane, Peripheral membrane protein, Intermembrane side (Potential). Cytoplasm. Note=The mitochondrial form is found in the intermembrane space probably attached to the outer side of the inner membrane.

KEGG Pathways
Map code	Pathways	E.C.
MAP00220	Urea cycle and metabolism of amino groups
MAP00260	Glycine, serine and threonine metabolism
MAP00330	Arginine and proline metabolism

Compound table
	Substrates		Products		Intermediates
KEGG-id	C00062	C00037	C00077	C00581
E.C.
Compound	L-Arginine	Glycine	L-Ornithine	Guanidinoacetate
Type	amino acids,amine group,imine group,lipid	amino acids	amino acids,amine group,lipid	amino acids,amine group,imine group
ChEBI	16467 16467	15428 57305 15428 57305	15729 15729	16344 57742 16344 57742
PubChem	28782 6322 28782 6322	5257127 750 5257127 750	6262 88747248 6262 88747248	3946848 4546993 763 3946848 4546993 763

1jdwA	Unbound	Unbound	Unbound	Unbound
1jdxA	Unbound	Unbound	Unbound	Unbound
2jdwA	Unbound	Unbound	Unbound	Unbound
2jdxA	Unbound	Unbound	Unbound	Unbound
3jdwA	Unbound	Unbound	Bound:ORN	Unbound
4jdwA	Bound:ARG	Unbound	Unbound	Unbound
5jdwA	Unbound	Bound:GLY	Unbound	Unbound
6jdwA	Unbound	Analogue:ABU	Unbound	Unbound
7jdwA	Unbound	Analogue:DAV	Unbound	Unbound
8jdwA	Unbound	Unbound	Unbound	Unbound
9jdwA	Unbound	Unbound	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
Swiss-prot;P50440 & literature [4]

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

1jdwA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407
1jdxA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407
2jdwA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407
2jdxA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407				deletion M302
3jdwA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407
4jdwA	ASP 170;ASP 254;HIS 303;ASP 305;				mutant C407A
5jdwA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407
6jdwA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407
7jdwA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407
8jdwA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407
9jdwA	ASP 170;ASP 254;HIS 303;ASP 305;CYS 407

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[2]	Fig.9	p.3380
[3]	p.196
[4]	p.488-489
[5]	Fig.8	p.17670-17671

References
[1]
Resource
Comments	ACTIVE SITE CYS-407
Medline ID	97220385
PubMed ID	9148748
Journal	Biochem J
Year	1997
Volume	322
Pages	771-6
Authors	Humm A, Fritsche E, Mann K, Gohl M, Huber R
Title	Recombinant expression and isolation of human L-arginine:glycine amidinotransferase and identification of its active-site cysteine residue.
Related PDB
Related UniProtKB	P50440
[2]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.9 ANGSTROMS) OF 64-423
Medline ID	97361819
PubMed ID	9218780
Journal	EMBO J
Year	1997
Volume	16
Pages	3373-85
Authors	Humm A, Fritsche E, Steinbacher S, Huber R
Title	Crystal structure and mechanism of human L-arginine:glycine amidinotransferase: a mitochondrial enzyme involved in creatine biosynthesis.
Related PDB	1jdw 2jdw 3jdw 4jdw
Related UniProtKB	P50440
[3]
Resource
Comments	REVIEW
Medline ID	97307728
PubMed ID	9165070
Journal	Biol Chem
Year	1997
Volume	378
Pages	193-7
Authors	Humm A, Fritsche E, Steinbacher S
Title	Structure and reaction mechanism of L-arginine:glycine amidinotransferase.
Related PDB
Related UniProtKB	P50440
[4]
Resource
Comments
Medline ID
PubMed ID	9266688
Journal	Eur J Biochem
Year	1997
Volume	247
Pages	483-90
Authors	Fritsche E, Humm A, Huber R
Title	Substrate binding and catalysis by L-arginine:glycine amidinotransferase--a mutagenesis and crystallographic study.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID	9922132
Journal	Biochemistry
Year	1998
Volume	37
Pages	17664-72
Authors	Fritsche E, Bergner A, Humm A, Piepersberg W, Huber R
Title	Crystal structure of L-arginine:inosamine-phosphate amidinotransferase StrB1 from Streptomyces griseus: an enzyme involved in streptomycin biosynthesis.
Related PDB
Related UniProtKB
[6]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (2.5 ANGSTROMS) OF 64-423
Medline ID	99115650
PubMed ID	9915841
Journal	J Biol Chem
Year	1999
Volume	274
Pages	3026-32
Authors	Fritsche E, Humm A, Huber R
Title	The ligand-induced structural changes of human L-Arginine:Glycine amidinotransferase. A mutational and crystallographic study.
Related PDB	1jdx 2jdx 5jdw 6jdw 7jdw 8jdw 9jdw
Related UniProtKB	P50440

Comments
The early papers, [2] & [3], proposed a catalytic mechanism as follows: The thiol group of Cys407, which plays a triple role as nucleophile, acid and base, makes a nucleophilic attack on the positively charged carbon atom of amidino group, and simultaneously protonates the leaving group, the epsilon-imino group of the arginine substrate. A tetrahedral intermediate is formed, which can be stabilized by the interaction between the guanidino nitrogen atoms and acidic residues, Asp170 and Asp305, and between the epsilon-imino nitrogen and His303. The bond between the epsilon-imino group and the amidino-carbon atom is broken. After the product of ornithine leaves the active site, another substrate, glycin can enter the site and bind to the amidino-carbon atom of the amidino-cysteine. The second half reaction may start with abstarction of proton from the positively charged glycine substrate by His303. The lone electron pair of the glycine nitorogen atom may make a nucleophilic attack at the amidino-carbon atom. In the final steps, formation of a tetrahedral intermediate and its breakdown by cleavage of the carbon-sulfur bond may occur. However, this second half reaction seemed to be speculative, according to the paper [2]. On the other hand, a more recent paper [4] revised this proposed mechanism. In the newly proposed mechanism, the reaction proceeds as follows: (1) Asp305 was suggested to act as a catalytic acid-base, by abstracting a thiol proton from the nucleophile, Cys407. (2) Cys407 makes a nucleophilic attack on the positively charged amidino carbon atom, leading to the formation of tetrahedral intermediate. (3) The tetrahedral intermediate can be stabilized by the interaction with Asp170 and Asp305. (4) In contrast, the sidechain of His303 seems to be protonated, activating the leaving group, the epsilon-imino group of arginine, as a general acid. (5) The breakdown of the tetrahedral intermediate generates the product, ornithine, and the amidino-Cys407. (6) A second substrate, glycine, binds to the active site as an acceptor group. (7) Probably, His303 acts as a general base, to activate the acceptor amine group of glycine. (8) The activated amine group makes a nucleophilic attack on the carbon atom of the planar amidino-Cys407. (9) Probably, Asp305 acts as a general acid to protonate the leaving Cys407, resulting the formation of the product, guanidino-acetic acid. (#) During the catalysis, Asp254 may be involved in the stablization and orientation of His303 towards the substrates (see [4]).

Comments

The early papers, [2] & [3], proposed a catalytic mechanism as follows:
The thiol group of Cys407, which plays a triple role as nucleophile, acid and base, makes a nucleophilic attack on the positively charged carbon atom of amidino group, and simultaneously protonates the leaving group, the epsilon-imino group of the arginine substrate.
A tetrahedral intermediate is formed, which can be stabilized by the interaction between the guanidino nitrogen atoms and acidic residues, Asp170 and Asp305, and between the epsilon-imino nitrogen and His303. The bond between the epsilon-imino group and the amidino-carbon atom is broken.
After the product of ornithine leaves the active site, another substrate, glycin can enter the site and bind to the amidino-carbon atom of the amidino-cysteine. The second half reaction may start with abstarction of proton from the positively charged glycine substrate by His303. The lone electron pair of the glycine nitorogen atom may make a nucleophilic attack at the amidino-carbon atom. In the final steps, formation of a tetrahedral intermediate and its breakdown by cleavage of the carbon-sulfur bond may occur. However, this second half reaction seemed to be speculative, according to the paper [2].
On the other hand, a more recent paper [4] revised this proposed mechanism. In the newly proposed mechanism, the reaction proceeds as follows:
(1) Asp305 was suggested to act as a catalytic acid-base, by abstracting a thiol proton from the nucleophile, Cys407.
(2) Cys407 makes a nucleophilic attack on the positively charged amidino carbon atom, leading to the formation of tetrahedral intermediate.
(3) The tetrahedral intermediate can be stabilized by the interaction with Asp170 and Asp305.
(4) In contrast, the sidechain of His303 seems to be protonated, activating the leaving group, the epsilon-imino group of arginine, as a general acid.
(5) The breakdown of the tetrahedral intermediate generates the product, ornithine, and the amidino-Cys407.
(6) A second substrate, glycine, binds to the active site as an acceptor group.
(7) Probably, His303 acts as a general base, to activate the acceptor amine group of glycine.
(8) The activated amine group makes a nucleophilic attack on the carbon atom of the planar amidino-Cys407.
(9) Probably, Asp305 acts as a general acid to protonate the leaving Cys407, resulting the formation of the product, guanidino-acetic acid.
(#) During the catalysis, Asp254 may be involved in the stablization and orientation of His303 towards the substrates (see [4]).

Created	Updated
2002-11-22	2009-02-26