Inhibitor Complexes of the <i>Pseudomonas</i> Serine-Carboxyl Proteinase

Wlodawer, Alexander; Li, Mi; Gustchina, Alla; Dauter, Zbigniew; Uchida, Kenichi; Oyama, Hiroshi; Goldfarb, Nathan E.; Dunn, Ben M.; Oda, Kōhei

doi:10.1021/bi011817n

Cited by 54 publications

(48 citation statements)

References 30 publications

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“…However, at the crystallization pH condition, pH 5.0, only Glu 272 and not Asp 276 is predicted to be deprotonated. The active site Glu is also found in dual conformations in some inhibitor complexes in structures of homologs crystallized at higher pH (25). In addition, the conformations of Glu 272 and Asp 276 in the glycosylated form of pro-TPP1 are similar to those found in mature enzymes of bacterial (27) is shown with ribbon depictions in gray and green, respectively.…”

Section: Resultsmentioning

confidence: 91%

“…The replacement of the His residue with the acidic residue Glu in the S53 family allows the active site serine to function in nucleophilic attack of substrate at acidic pH (14), with the Glu abstracting the hydroxyl proton from the serine (14,(23)(24)(25)(26)34). In addition, there is a second conserved Asp residue (Asp 360 ), designated here as DЈ, in place of a conserved Asn in the subtilisin family.…”

Section: Resultsmentioning

confidence: 99%

“…21), which includes a number of unusual bacterial serine peptidases (22). High resolution crystal structures of both free and inhibitor-bound complexes have been determined for three bacterial members of this family (sedolisin (23)(24)(25)(26), kumamolisin (27,28), and kumamolisin-As (29,30)), and for one (kumamolisin), the structure of a mutant, inactive precursor form has also been obtained (28). These proteins share a common subtilisin-like fold, an octahedrally coordinated calcium-binding site, and an active site that contains an unusual Ser-Glu-Asp (SED) catalytic triad, rather than the SerHis-Asp (SHD) triad of subtilisin (31,32).…”

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confidence: 99%

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Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

Guhaniyogi

Sohár²,

Das

et al. 2009

Journal of Biological Chemistry

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Late infantile neuronal ceroid lipofuscinosis is a fatal childhood neurological disorder caused by a deficiency in the lysosomal protease tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian member of the S53 family of serine proteases, a group characterized by a subtilisin-like fold, a SerGlu-Asp catalytic triad, and an acidic pH optimum. TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is proteolytically processed into the active enzyme after exposure to low pH in vitro or targeting to the lysosome in vivo. In this study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing four Asn-linked N-acetylglucosamines that is indistinguishable from fully glycosylated TPP1 in terms of autocatalytic processing of the proform and enzymatic properties of the mature protease. The crystal structure of deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large 151-residue C-shaped prodomain makes extensive contacts as it wraps around the surface of the catalytic domain with the two domains connected by a 24-residue flexible linker that passes through the substrate-binding groove. The proenzyme structure reveals suboptimal catalytic triad geometry with its propiece linker partially blocking the substrate-binding site, which together serve to prevent premature activation of the protease. Finally, we have identified numerous processing intermediates and propose a structural model that explains the pathway for TPP1 activation in vitro. These data provide new insights into TPP1 function and represent a valuable resource for constructing improved TPP1 variants for treatment of late infantile neuronal ceroid lipofuscinosis.

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Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

Guhaniyogi

Sohár²,

Das

et al. 2009

Journal of Biological Chemistry

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“…However, these proteases are not inactivated by pepstatin, which is a natural inhibitor of the aspartic acid class of proteases. Rather they are inactivated by inhibitors with aldehyde functional groups such as chymostatin, which typically modify the serine nucleophile within serine proteases (Wlodawer et al 2001b). …”

Section: Ser/glu/asp Triad: Sedolisin Proteasesmentioning

confidence: 99%

Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

2008

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Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called ''classical'' catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of ''nonclassical'' serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/ His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.

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“…The average active site structure for the kumamolisin-Assubstrate (GPH*FF) complex obtained from the QM/MM MD simulations is given in Figure 1A. Ser278 is the nucleophile that attacks the carbonyl carbon atom (C) of the substrate, while Glu78 and Asp164 act as the general base and acid catalysts, respectively (4)(5)(6)(7)(8)(9)(10)(15)(16)(17). Two additional residues, Glu32 and Trp129, interact with Asp82 of the catalytic triad through hydrogen-bonding networks.…”

Section: Structural and Dynamic Properties Of The Substratementioning

confidence: 99%

The QM/MM Molecular Dynamics and Free Energy Simulations of the Acylation Reaction Catalyzed by the Serine-Carboxyl Peptidase Kumamolisin-As

Guo

Wlodawer

et al. 2007

Biochemistry

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Quantum mechanical/molecular mechanical molecular dynamics and free energy simulations are performed to study the acylation reaction catalyzed by kumamolisin-As, a serine-carboxyl peptidase, and to elucidate the catalytic mechanism and the origin of substrate specificity. It is demonstrated that the nucleophilic attack by the serine residue on the substrate may not be the rate-limiting step for the acylation of the GPH*FF substrate. The present study also confirms the earlier suggestions that Asp164 acts as a general acid during the catalysis and that the electrostatic oxyanion hole interactions may not be sufficient to lead a stable tetrahedral intermediate along the reaction pathway. Moreover, Asp164 is found to act as a general base during the formation of the acyl-enzyme from the tetrahedral intermediate. The role of dynamic substrate assisted catalysis (DSAC) involving His at the P 1 site of the substrate is examined for the acylation reaction. It is demonstrated that the bond-breaking and -making events at each stage of the reaction trigger a change of the position for the His side chain and lead to the formation of the alternative hydrogen bonds. The back and forth movements of the His side chain between the CdO group of Pro at P 2 and O δ2 of Asp164 in a ping-pong-like mechanism and the formation of the alternative hydrogen bonds effectively lower the free energy barriers for both the nucleophilic attack and the acyl-enzyme formation and may therefore contribute to the relatively high activity of kumamolisin-As toward the substrates with His at the P 1 site.Kumamolsin-As belongs to the recently characterized family of serine-carboxyl peptidases (sedolisins) that were originally described by Murao, Oda, and co-workers about 20 years ago (1-3). Sedolisins are present in a wide variety of organisms, including archaea, bacteria, molds, slime molds (mixomycetes), amoebas, fishes, and mammals, and they are active at low pH and often high temperature. This family of enzymes seems to have been derived through divergent evolution from a common ancestor with classical serine peptidases; the structural studies (4-10) showed that sedolisins have a fold resembling that of subtilisin, although they are significantly larger (the mature catalytic domains contain approximately 375 amino acid residues). One interesting question is why nature needs another family of subtilisin-like peptidases. For bacterial sedolisins, one possible explanation is that some organisms might have to utilize such evolved serine-carboxyl peptidases for their survival, presumably under acidic environments and high temperature.The biological importance of sedolisins in mammals has already been demonstrated by the fact that mutations leading to the loss of the human enzyme CLN2 (11-13), another member of the sedolisin family, result in a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis (14).The three-dimensional structures are available for three members of the sedolisin family, including sedolisin...

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Inhibitor Complexes of the Pseudomonas Serine-Carboxyl Proteinase

Cited by 54 publications

References 30 publications

Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

The QM/MM Molecular Dynamics and Free Energy Simulations of the Acylation Reaction Catalyzed by the Serine-Carboxyl Peptidase Kumamolisin-As

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