Energetics of folding subtilisin BPN'

Bryan, Philip N.; Alexander, Patrick; Strausberg, S; Schwarz, Frederick P.; Lan, Wenxian; Gilliland, Gary L.; Gallagher, D.T.

doi:10.1021/bi00136a003

Cited by 106 publications

(122 citation statements)

References 38 publications

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“…Sites 1 and 2 represent high-and low-affinity binding sites, respectively. The enzyme is greatly destabilized with respect to both thermal denaturation and autodegradation and is thereby inactivated upon removal of Ca 2ϩ from site 1 (11,42,59). However, pro-subtilisin E has been reported to be folded and autoprocessed in the absence of Ca 2ϩ (66).…”

Section: Discussionmentioning

confidence: 99%

Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Pulido

Saito

Tanaka

et al. 2006

Appl Environ Microbiol

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Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca 2؉ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca 2؉ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin‫)ء‬ but forms an inactive complex with T. kodakaraensis mat-subtilisin‫,ء‬ at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin‫,ء‬ and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca 2؉ but was activated upon incubation with Ca 2؉ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin‫,ء‬ indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ϳ5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin‫ء‬ and noncompetitively inhibited its activity with a K i of 25 ؎ 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.Subtilisin-like serine proteases (subtilases) are widely distributed in various organisms, including bacteria, archaea, and eucaryotes (48). They are divided into six families (47). Of these, the structures and functions of the subtilisin family (EC 3.4.21.108), which is represented by subtilisin E from Bacillus subtilis (52), subtilisin BPNЈ from Bacillus amyloliquefaciens (61), and subtilisin Carlsberg from Bacillus licheniformis (25), have been most extensively studied. The crystal structures of these subtilisins have been determined (26,54,64). Because subtilisins are commercially valuable enzymes, extensive attempts to improve their activities and stabilities with proteinengineering technology have also been made (10, 55, 60). The subtilisin family includes subtilisins from (hyper)thermophiles (13,27,28,35) and psychrophiles (3,14,29,37). The crystal structures of some of them have been determined (2,4,50,57). These thermostable and thermolabile subtilisins have been regarded not only as good models for studying stability-activitystructure relationships of proteins, but also as potent...

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Section: Discussionmentioning

confidence: 99%

Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Pulido

Saito

Tanaka

et al. 2006

Appl Environ Microbiol

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“…Numerous sitespecific mutations that increase thermostability have been described (Pantoliano et al, 1989;Gilliland, Gallagher & Bryan, 1996). The stronger of SBT's two calcium-binding sites binds Ca 2+ with K, = 7 x 10°M -~, and is important in folding and stability (Bryan et al, 1992). The other site is weaker and less specific, consisting of two overlapping subsites for monovalent and divalent cations (Pantoliano et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

Subtilisin BPN' at 1.6 Å Resolution: Analysis for Discrete Disorder and Comparison of Crystal Forms

Gallagher¹,

Oliver²,

Bott³

et al. 1996

Acta Cryst D

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The three-dimensional structure of the serine protease subtilisin BPN' (SBT) has been refined at 1.6A resolution in space group C2 to a final R value of 0.17. 17 regions of discrete disorder have been identified and analyzed. Two of these are dualconformation peptide units; the remainder invclve alternate rotamers of side chains either alone o~ in small clusters. The structure is compared with previously reported high-resolution models of SBT in two other space groups, P212~2 ~ and P2~. Apart from the surface, there are no significant variations in structure among the three crystal forms. Structural variations observed at the protein surface occur predominantly in regions of protein-protein contact. The crystal packing arrangements in the three space groups are compared.

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“…In most cases propeptides are also indispensable for correct folding of the enzymes (8). In some enzymes folding of the mature form is extremely slow, and the propeptide assists in overcoming the kinetic barrier (9), which may also be overcome by physicochemical factors such as high ionic strength in subtilisin, for example (10). Proenzymes are quite often more stable than mature enzymes (11,12) and can represent a pool of latent enzyme until the activation occurs in the proper conditions.…”

mentioning

confidence: 99%

pH-induced Conformational Transitions of the Propeptide of Human Cathepsin L

Jerala

Žerovnik

Kidrič

et al. 1998

Journal of Biological Chemistry

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Synthesis of proteases as inactive zymogens is a very important mechanism for the regulation of their activity. For lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the propeptide of cathepsin L loses most of the tertiary structure, but almost none of the secondary structure is lost. Another partially structured intermediate, prone to aggregation, was identified between pH 6.5 and 4. The conformation, populated below pH 4, where the activation of cathepsin L occurs, is not completely unfolded and has the properties of molten globule, including characteristic binding of the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the propeptide parallels a decrease of its affinity for cathepsin L and suggests the mechanism for the acidic zymogen activation. Addition of anionic polysaccharides that activate cathepsin L already at pH 5.5 unfolds the tertiary structure of the propeptide at this pH. Propeptide of human cathepsin L which is able to fold independently represents an evolutionary intermediate in the emergence of novel inhibitors originating from the enzyme proregions.All lysosomal and most other proteases are synthesized in the form of inactive precursors (1, 2). Propeptides are generally located N-terminal to the mature enzyme, and activation of the enzyme is accomplished by cis-or trans-cleavage of the propeptide. Propeptides vary from a few (e.g. trypsin) to more than 200 residues (e.g. cathepsin C). Longer propeptides are generally strong and specific inhibitors of their mature enzymes (3-7). In most cases propeptides are also indispensable for correct folding of the enzymes (8). In some enzymes folding of the mature form is extremely slow, and the propeptide assists in overcoming the kinetic barrier (9), which may also be overcome by physicochemical factors such as high ionic strength in subtilisin, for example (10). Proenzymes are quite often more stable than mature enzymes (11, 12) and can represent a pool of latent enzyme until the activation occurs in the proper conditions. Propeptides are also involved in targeting to specific organelles (13, 14); they can affect posttranslational modification such as glycosylation (15) and mediate interactions with other molecules (16,17). Propeptides can be cleaved either by other proteases or by intra-or intermolecular autocatalysis. pH change is one of the most common environmental parameters responsible for triggering the activation of proteases, occurring in cysteine, aspartic acid, and metalloproteases (1,18,19). Low pH is thought either to increase the susceptibility of the propeptide as a substrate due to the protonation of groups close to the cleavage site or to cause a conformational change in the propeptide or enzyme.Cathepsin L is one of the most active cysteine proteases and accounts for most of the lysosomal cysteine protease activity (20). It has been implicated in a range of processes incl...

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Energetics of folding subtilisin BPN'

Cited by 106 publications

References 38 publications

Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Subtilisin BPN' at 1.6 Å Resolution: Analysis for Discrete Disorder and Comparison of Crystal Forms

pH-induced Conformational Transitions of the Propeptide of Human Cathepsin L

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Energetics of folding subtilisin BPN'

Cited by 106 publications

References 38 publications

Ca 2+ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Ca 2+ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Subtilisin BPN' at 1.6 Å Resolution: Analysis for Discrete Disorder and Comparison of Crystal Forms

pH-induced Conformational Transitions of the Propeptide of Human Cathepsin L

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Product

Resources

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Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding