The structure of PAS shows that the resting state of the key catalytic residue in sulfatases is a formylglycine hydrate. These structural data establish a mechanism for sulfate ester cleavage involving an aldehyde hydrate as the functional group that initiates the reaction through a nucleophilic attack on the sulfur atom in the substrate. The alcohol is eliminated from a reaction intermediate containing pentacoordinated sulfur. Subsequent elimination of the sulfate regenerates the aldehyde, which is again hydrated. The metal cation involved in stabilizing the charge and anchoring the substrate during catalysis is established as calcium.

Arylsulfatase A belongs to the sulfatase family whose members carry a C␣-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members Mammalian sulfatases are involved in the degradation of sulfated substrates like mucopolysaccharides, cerebroside sulfates, and sulfated steroids. The key residue for catalytic activity in sulfate ester cleavage has recently been shown to be a C␣-formylglycine (FGly; Refs. 1 and 2), 1 which is post-translationally generated from a cysteine in the endoplasmic reticulum (3). Failure of this amino acid modification leaves newly synthesized sulfatases inactive and is the cause of multiple sulfatase deficiency, a rare but fatal lysosomal storage disorder (Ref. 1; reviewed in Refs. 4 and 5). X-ray determination of the three-dimensional structures of two human sulfatases, arylsulfatase A (ASA; Ref. 6) and N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB; Ref. 7), revealed that the FGly residue is buried at the bottom of a cavity that is formed by positively and negatively charged amino acids. This cavity proved to be the active site by two lines of evidence. Cocrystallization of ASB with vanadate, a potent inhibitor of ASB, revealed that vanadate was covalently bound to FGly (7). In addition, an ASA mutant containing serine instead of FGly was found to be covalently sulfated at the position of FGly 69 after incubation with sulfate ester (see below and Ref. 8). The 2-fold disordered electron density of FGly was interpreted to be an aldehyde hydrate with two geminal hydroxyls protruding into the lumen of the cavity (6). In close vicinity to the aldehyde hydrate a metal ion (Mg 2ϩ in ASA and Ca 2ϩ in ASB) is complexed by three aspartates and one asparagine. Two lysines, two histidines, and a serine complete the arrangement of residues potentially involved in binding and cleavage of the sulfate group within the active site.These data led to a proposal for the catalytic mechanism of sulfate ester cleavage (6) as follows (Fig. 1). In the first halfcycle, one of the two geminal oxygens of the aldehyde hydrate attacks the sulfur of the sulfate ester leading to a transesterification of the sulfate group onto the aldehyde hydrate. Simultaneously the substrate alcohol is released. In the second halfcycle, sulfate is eliminated from the enzyme-sulfate intermediate by an intramolecular rearrangement induced by the second oxygen. By this "intramolecular hydrolysis" the aldehyde group is regenerated. The essential role of FGly in forming the transient enzyme-sulfate ester has been shown indire...

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In one of the most common mutations causing metachromatic leukodystrophy, the P426L-allele of arylsulfatase A (ASA), the deficiency of ASA results from its instability in lysosomes. Inhibition of lysosomal cysteine proteinases protects the P426L-ASA and restores the sulfatide catabolism in fibroblasts of the patients. P426L-ASA, but not wild type ASA, was cleaved by purified cathepsin L at threonine 421 yielding 54-and 9-kDa fragments. X-ray crystallography at 2.5-Å resolution showed that cleavage is not due to a difference in the protein fold that would expose the peptide bond following threonine 421 to proteases. Octamerization, which depends on protonation of Glu-424, was impaired for P426L-ASA. The mutation lowers the pH for the octamer/ dimer equilibrium by 0.6 pH units from pH 5.8 to 5. Arylsulfatase A (ASA) 1 is a lysosomal enzyme that is synthesized as a 52-kDa polypeptide. In the endoplasmic reticulum ASA receives three N-linked oligosaccharides, undergoes con-

Improved data quality now makes it feasible to exploit the weak anomalous signal derived only from the sulfurs inherent to the protein or in particular from halide ions incorporated by soaking. The latter technique requires the location of a high number of partially occupied halide sites. This number appears to be roughly proportional to the exposed protein surface. This paper explores the application of dual-space ab initio methods as implemented in the program SHELXD to the location of substructures of sulfur in SAD experiments, bromide in SAD and MAD experiments and iodide using SAD and SIRAS to determine the anomalous-atom substructure. Sets of atoms consistent with the Patterson function were generated as a starting point for the dual-space recycling procedure in SHELXD. The substructure is then expanded to the full structure by maximum-likelihood phasing with SHARP and density modification with the program DM. Success in the location of the substructures and subsequent phasing depends critically on the quality of the data and on the extent of the anomalous signal. This varies with each crystal and soak, but for the same crystal the significance of the anomalous signal was found to be highly sensitive to the redundancy of the intensity measurements, which in some cases made all the difference. This is illustrated by the determination of the previously unknown structure of repeat 11 of the human mannose-6-phosphate/insulin-like growth factor II receptor (Man6P/IGFII-receptor), with 310 amino acids in the asymmetric unit, which was phased by soaking the crystals in a cryoprotectant solution containing halide anions.