Functional analyses of active site residues of human lysosomal aspartylglucosaminidase: implications for catalytic mechanism and autocatalytic activation.

Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved HisAsp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-Å resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature.

Section: Glycosylasparaginase (Ga)mentioning

confidence: 99%

Two-step Dimerization for Autoproteolysis to Activate Glycosylasparaginase

Wang

Guo

2003

“…On the other hand, mutagenesis studies of aspartylglucosaminidases have established that substitution of the equivalent of Thr 230 by Ala does not abolish autoproteolysis, although it makes it less efficient (19,47). Moreover, it was reported that although this mutation completely inhibits the catalytic activity of the human enzyme (47), the bacterial homolog retains 30% of its original activity (19).…”

Section: Conformation Of Thr 179 -A Theoretically Modeled Thrmentioning

confidence: 99%

“…As a conclusion, it has been postulated that in aspartylglucosaminidases, the Thr residue is required for proper positioning of Asp 151 (20). With regard to Thr 197 , an alanine substitution of its equivalent in aspartylglucosaminidase resulted in a processing-defective human enzyme (47), whereas for the bacterial protein, only the processing rate was affected, whereas the reaction product was unchanged (19). Those observations suggest that none of these Thr residues is a universal and absolute prerequisite for the autoproteolytic process.…”

Section: Conformation Of Thr 179 -A Theoretically Modeled Thrmentioning

confidence: 99%

The Mechanism of Autocatalytic Activation of Plant-type L-Asparaginases

Michalska

Hernández-Santoyo

Jaskólski

2008

Plant L-asparaginases and their bacterial homologs, such as EcAIII found in Escherichia coli, form a subgroup of the N-terminal nucleophile (Ntn)-hydrolase family. In common with all Ntn-hydrolases, they are expressed as inactive precursors that undergo activation in an autocatalytic manner. The maturation process involves intramolecular hydrolysis of a single peptide bond, leading to the formation of two subunits (␣ and ␤) folded as one structural domain, with the nucleophilic Thr residue located at the freed N terminus of subunit ␤. The mechanism of the autocleavage reaction remains obscure. We have determined the crystal structure of an active site mutant of EcAIII, with the catalytic Thr residue substituted by Ala (T179A). The modification has led to a correctly folded but unprocessed molecule, revealing the geometry and molecular environment of the scissile peptide bond. The autocatalytic reaction is analyzed from the point of view of the Thr 179 side chain rotation, identification of a potential general base residue, and the architecture of the oxyanion hole.Posttranslational modifications of proteins can be divided into reactions leading to covalent attachment, usually at a side chain, of a specific chemical group, such as phosphate or carbohydrate, and into reactions that lead to cleavage or cleavage/ rearrangement of the polypeptide backbone. The latter processes can be enzyme-catalyzed or occur without an additional biocatalyst. An important class of backbone rearrangement is connected with autocatalytic processes in maturating proteins. Notable examples in this category include intein splicing and simple backbone cleavage. Protein splicing requires cleavage of two peptide bonds that surround the so-called intein, followed by ligation of the flanking polypeptides, the N-and C-exteins. The process consists of four steps: acyl rearrangement, transesterification, cyclization of an asparagine residue, and a second acyl rearrangement (1). In maturation following the autocleavage pathway, only a single peptide cleavage occurs, and the mechanism includes only acylation and water-dependent deacylation. Although the mechanism of protein splicing has been extensively studied and seems to be well understood, the autoproteolysis reactions are rather obscure. The known examples of autoproteolytic proteins include a large group of N-terminal nucleophile (Ntn) 2 -hydrolases (2). In Ntn enzymes, a cleavage of a precursor molecule is required to generate a catalytic residue at the newly formed N terminus, which can be a threonine, serine, or cysteine. The family of Ntn-hydrolases includes such enzymes as aspartylglucosaminidases (3, 4), penicillin acylases (5-7), taspase1 (8), and plant-type L-asparaginases (9 -12). Sequence similarity within the Ntn-hydrolase family is very limited, but despite the variation of primary structure, the proteins share a common sandwich-like ␣␤␤␣ fold created by two ␤-sheets surrounded by two layers of ␣-helices (13). The autocatalyzed maturation of Ntn-hydrolases involves either the remo...

“…Bound water enhances the nucleophilicity of the Thr O␥ at the ϩ1 position in the PS precursor (4), whereas the Asp-151 carboxylate at the Ϫ1 position promotes the nucleophilicity of the Thr-152 at the ϩ1 position in the GA precursor (7). Consequently, the hydroxyl of the catalytic Thr attacks the adjacent scissile peptide bond to carry out intramolecular cleavage (4,(7)(8)(9)(10).…”

Section: Conformation Of the F177␤p Precursor Cad Forces The Eliminatmentioning

confidence: 99%

A Bound Water Molecule Is Crucial in Initiating Autocatalytic Precursor Activation in an N-terminal Hydrolase

Yoon

Kim

et al. 2004

Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an ␣-subunit, a spacer sequence, and a ␤-subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with ␣-and ␤-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1␤ onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1␤ to generate a strong nucleophile and to direct the OG atom of the Ser-1␤ to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme.Cephalosporin acylase (CA) 1 is a new family of the N-terminal (Ntn) hydrolase superfamily that is defined by the SCOP data base (1). The Ntn hydrolase superfamily is defined by SCOP as a motif containing four layers of ␣-helices and ␤-sheets in a ␣␤␤␣ fashion. There are five known families of the Ntn hydrolase superfamily; they are the class II glutamine amidotransferases, penicillin G acylase, penicillin V acylase, proteasome ␤ subunit, and glycosyl asparaginase (1).Ntn hydrolases are generally translated into a precursor enzyme. Several Ntn hydrolases are activated through autoproteolytic processing by the Ser, Thr, or Cys residues. Autoproteolytic processing occurs in an intramolecular manner in several Ntn hydrolases. These include glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase, penicillin G acylase, proteasome ␤ subunit (PS), glycosyl asparaginase (GA), and cephalosporin acylase (2-4).The gene structure of the open reading frame of CAs generally consists of a signal peptide that is followed by an ␣-subunit, a spacer sequence, and a ␤-subunit, all of which are translated into a single polypeptide chain, the CA precursor. The precursor is post-translationally modified into an active heterodimeric enzyme with ␣ and ␤ subunits, first by intramolecular cleavage process and, second, by intermolecular cleavage process.The heterodimeric enzyme is envisaged from the structural studies of the active CA and precursor CA, in which the nascent polypeptide (precursor) of cephalosporin acylase is autoproteolytically activat...