Identification of a Glutamic Acid and an Aspartic Acid Residue Essential for Catalytic Activity of Aspergillopepsin II, a Non-pepsin Type Acid Proteinase

Huang, Xiangping; Kagami, Naofumi; Inoue, Hiroo; Kojima, Masaki; Kimura, Takao; Makabe, Osamu; Suzuki, Koichi; Takahashi, Kenji

doi:10.1074/jbc.m910243199

Cited by 25 publications

(23 citation statements)

References 35 publications

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“…The results may also indicate that there are subtle differences in contribution by respective residues between stabilization and inhibition. The denaturation curves in the presence of the propeptides, Glu 12 -Lys 31 and Lys 18 -Tyr 25 , showed biphasic transition (Fig. 5, C and D), consistent with the specific binding of these peptides to the enzyme.…”

Section: Thermal Stabilization Of Agp By Truncated Propeptides and Almentioning

confidence: 71%

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Kubota

Nishii

Kojima

et al. 2005

Journal of Biological Chemistry

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Aspergilloglutamic peptidase (formerly called aspergillopepsin II) is an acid endopeptidase produced byAspergillus niger var. macrosporus, with a novel catalytic dyad of a glutamic acid and a glutamine residue, thus belonging to a novel peptidase family G1. The mature enzyme is generated from its precursor by removal of the putative 41-residue propeptide and an 11-residue intervening peptide through autocatalytic activation. In the present study, the propeptide (Ala 1 -Asn 41 ) and a series of its truncated peptides were chemically synthesized, and their effects on the enzyme activity and thermal stability were examined to identify the sequences and residues in the propeptide most critical to the inhibition and thermal stabilization. The synthetic propeptide was shown to be a potent competitive inhibitor of the enzyme (K i ‫؍‬ 27 nM at pH 4. Propeptides are known to be present in many protein precursors mostly at their N termini and are assumed to play various roles in the proteins such as inhibition of protein functions, stabilization of precursor structures, promotion of polypeptide folding, and/or intercellular protein sorting and targeting (1-6). These roles, however, have not yet been fully understood, and further studies on various proteins are necessary. As for the inhibitory properties of the propeptides of peptidases, there are numbers of reports describing their inhibition profiles and type of inhibition (7-25). However, only a few attempts have been made so far to identify critical sequences and/or residues in the propeptide important for inhibition of the corresponding mature peptidase, and much of the inhibition mechanisms, the structural determinants in the propeptide, and the sites of binding involved in the inhibition remains to be established. On the other hand, there are few studies that analyzed the effects of propeptide and its truncated fragments on the stability of the mature enzyme to identify critical sequences and/or residues contributing to the enzyme stability.Aspergilloglutamic peptidase (AGP 1 ; MEROPS ID: G01.002; formerly called aspergillopepsin II) is a pepstatin-insensitive acid endopeptidase produced by the fungus Aspergillus niger var. macrosporus (26). Like scytalidoglutamic peptidase (or eqolisin; MEROPS ID: G01.001; formerly called scytalidopepsin B) (27, 28), AGP also belongs to the newly established family of glutamic peptidases (i.e. peptidase family G1). The enzyme has two essential residues, a glutamic acid and a glutamine residue, at the active site (29 -32), which is thought to form a catalytic dyad, and is not inhibited by any of the known inhibitors for aspartic, cysteine, metallo-, and serine peptidases. The enzyme is synthesized as a 282-residue preproenzyme, and after removal of the 18-residue putative signal peptide, the proform is converted autocatalytically to the twochain mature enzyme (composed of a 39-residue light chain and a 173-residue heavy chain) by liberation of the putative 41-residue propeptide (Ala 1 -Asn 41 ) and the 11-residue intervening peptide...

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Section: Thermal Stabilization Of Agp By Truncated Propeptides and Almentioning

confidence: 71%

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Kubota

Nishii

Kojima

et al. 2005

Journal of Biological Chemistry

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“…The calculated molecular mass of the mature form is 21.37 kDa, which is identical to the value obtained by MALDI-TOF MS. Protein alignment studies indicate that mature TGP1 shares 64 and 47% identity with glutamic peptidases from A. niger (AGP) (26) and Scytalidium lignocolum (SGP) (27), respectively, and the active site dyad is proposed by homology to be Gln 116 and Glu 201 (Fig. 2).…”

Section: Pep1mentioning

confidence: 99%

Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the Fungus Talaromyces emersonii

O’Donoghue

Mahon

Goetz

et al. 2008

Journal of Biological Chemistry

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The thermophilic filamentous fungus Talaromyces emersonii secretes a variety of hydrolytic enzymes that are of interest for processing of biomass into fuel. Many carbohydrases have been isolated and characterized from this fungus, but no studies had been performed on peptidases. In this study, two acid-acting endopeptidases were isolated and characterized from the culture filtrate of T. emersonii. One of these enzymes was identified as a member of the recently classified glutamic peptidase family and was subsequently named T. emersonii glutamic peptidase 1 (TGP1). The second enzyme was identified as an aspartyl peptidase (PEP1). TGP1 was cloned and sequenced and shown to exhibit 64 and 47% protein identity to peptidases from Aspergillus niger and Scytalidium lignocolum, respectively. Substrate profiling of 16 peptides determined that TGP1 has broad specificity with a preference for large residues in the P1 site, particularly Met, Gln, Phe, Lys, Glu, and small amino acids at P1 such as Ala, Gly, Ser, or Thr. This enzyme efficiently cleaves an internally quenched fluorescent substrate containing the zymogen activation sequence (k cat /K m ‫؍‬ 2 ؋ 10 5 M ؊1 s ؊1 ). Maximum hydrolysis occurs at pH 3.4 and 50°C. The reaction is strongly inhibited by a transition state peptide analog, TA1 (K i ‫؍‬ 1.5 nM), as well as a portion of the propeptide sequence, PT1 (K i ‫؍‬ 32 nM). Ex vivo studies show that hyphal extension of T. emersonii in complex media is unaffected by the aspartyl peptidase inhibitor pepstatin but is inhibited by TA1 and PT1. This study provides insight into the functional role of the glutamic peptidase TGP1 for growth of T. emersonii.As chemoheterotrophs, filamentous fungi secrete a variety of polymer-degrading hydrolases such as peptidases and carbohydrases that degrade organic material in the local environment to provide essential nutrients for the growing hyphae. Many fungi release organic acids to acidify the environment while secreting enzymes that are optimally active under these conditions. Traditionally, acid-acting fungal peptidases were assigned to the aspartic protease family and include aspergillopepsin from various Aspergillus species (1) and penicillopepsin from Penicillium species (2). These enzymes have two active-site aspartic acid residues and are strongly inhibited by pepstatin. Recently, two distinct groups of acid peptidases were identified that are insensitive to pepstatin and lack the catalytic motif observed in pepsintype peptidases. One group, "sedolisins" possess a fold similar to members of the subtilisin family but contain an active site triad of serine (S), glutamic acid (E), and aspartic acid (D) (3). They are sensitive to leupeptin and have homologs in bacteria (4), fungi (5), slime mold (6), and mammals (7). The second group of pepstatin-insensitive acid peptidases share a distinct set of structural, enzymatic, and physicochemical properties, and to date have only been identified in a select group of fungi (8). Members of this novel family possess a catalytic dyad consis...

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“…Glu136 and Gln53 were identified as the presumed catalytic residues by structural analysis [9]. Asp43 which corresponds to the Asp53 in ANCP, had been suggested as one of the catalytic residues by site-directed mutagenesis of ANCP [16].…”

Section: Identification Of Catalytic Dyad Glu136 and Gln53mentioning

confidence: 99%

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

et al. 2005

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Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P 3 (basic amino acid), P 0 1 (small a.a.), and P 0 3 (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. k cat , K m , andwere 34.8 s À1 , 0.065 lM, and 535 lM À1 s À1 , respectively. K i of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH 2 for SGP was 1.2 · 10 À10 M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.

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Identification of a Glutamic Acid and an Aspartic Acid Residue Essential for Catalytic Activity of Aspergillopepsin II, a Non-pepsin Type Acid Proteinase

Cited by 25 publications

References 35 publications

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the Fungus Talaromyces emersonii

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

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