PDBsum entry 1e2s

Hydrolase

PDB id: 1e2s

Contents

Protein chain

481 a.a. *

Ligands

NAG-NAG

CSN

Metals

_MG

Waters ×166

* Residue conservation analysis

PDB id:

1e2s

Name:

Hydrolase

Title:

Crystal structure of an arylsulfatase a mutant c69a

Structure:

Arylsulfatase a. Chain: p. Synonym: asa,cerebroside-sulfatase. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Organ: testis. Cellular_location: lysosome. Gene: arsa. Expressed in: mus musculus. Expression_system_taxid: 10090. Expression_system_cell_line: mef

Biol. unit:

Homo-Dimer (from PDB file)

Resolution:

2.35Å R-factor: 0.194 R-free: 0.236

Authors:

R.Von Buelow,B.Schmidt,T.Dierks,K.Von Figura,I.Uson

Key ref:

R.von Bülow et al. (2001). Crystal structure of an enzyme-substrate complex provides insight into the interaction between human arylsulfatase A and its substrates during catalysis. J Mol Biol, 305, 269-277. PubMed id: 11124905

Date:

24-May-00 Release date: 06-Dec-00

Protein chain

P15289  (ARSA_HUMAN) -  Arylsulfatase A from Homo sapiens

Seq: 507 a.a.

Struc: 481 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.1.6.8 - cerebroside-sulfatase.
• Reaction: an N-acyl-1-beta-D-(3-O-sulfo)-galactosyl-sphing-4-enine + H2O = a beta- D-galactosyl-(1<->1')-N-acylsphing-4-enine + sulfate + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Key reference

J Mol Biol 305:269-277 (2001)

PubMed id: 11124905

Crystal structure of an enzyme-substrate complex provides insight into the interaction between human arylsulfatase A and its substrates during catalysis.

R.von Bülow, B.Schmidt, T.Dierks, K.von Figura, I.Usón.

ABSTRACT

Arylsulfatase A (ASA) belongs to the sulfatase family whose members carry a C(alpha)-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The crystal structures of two arylsulfatases, ASA and ASB, and kinetic studies on ASA mutants led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters. The structures of two ASA mutants that lack the functional C(alpha)-formylglycine residue 69, in complex with a synthetic substrate, have been determined in order to unravel the reaction mechanism. The crystal structure of the inactive mutant C69A-ASA in complex with p-nitrocatechol sulfate (pNCS) mimics a reaction intermediate during sulfate ester hydrolysis by the active enzyme, without the covalent bond to the key side-chain FGly69. The structure shows that the side-chains of lysine 123, lysine 302, serine 150, histidine 229, the main-chain of the key residue 69 and the divalent cation in the active center are involved in sulfate binding. It is proposed that histidine 229 protonates the leaving alcoholate after hydrolysis.C69S-ASA is able to bind covalently to the substrate and hydrolyze it, but is unable to release the resulting sulfate. Nevertheless, the resulting sulfation is low. The structure of C69S-ASA shows the serine side-chain in a single conformation, turned away from the position a substrate occupies in the complex. This suggests that the double conformation observed in the structure of wild-type ASA is more likely to correspond to a formylglycine hydrate than to a twofold disordered aldehyde oxo group, and accounts for the relative inertness of the C69S-ASA mutant. In the C69S-ASA-pNCS complex, the substrate occupies the same position as in the C69A-ASA-pNCS complex, which corresponds to the non-covalently bonded substrate. Based on the structural data, a detailed mechanism for sulfate ester cleavage is proposed, involving an aldehyde hydrate as the functional group.