Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching

Grøn, Hanne; Meldal, Morten; Breddam, Klaus

doi:10.1021/bi00141a008

Cited by 142 publications

(70 citation statements)

References 31 publications

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“…While the hydrophobic S I and S4 binding pockets appear to dominate substrate binding in subtilisins (Gron et al, 1992), our analysis of binding sites in NisP suggests that electrostatic interactions may alternatively contribute to binding and even dominate overall selectivity (or specificity). In particular, in the case of NisP and EpiP, one or more negative charges in the S I pocket can lead to a high selectivity for a basic PI residue in the substrate.…”

Section: Discussionmentioning

confidence: 77%

“…The S4 pocket of NisP is predicted to be smaller than that of subtilisin due to several large predominantly hydrophobic side chains (Table II). While interaction of the small residue Ala(P4) of precursor nisin in this pocket may be less important than the S I-P I interaction, it was shown that Ala(P4) is not unfavourable for subtilisin and is still a good substrate even for the larger hydrophobic S4 pocket of subtilisin (Gron et al, 1992). The Ser(P3) of precursor nisin does not appear to interact directly with NisP, although hydrogen bonding to NI43 is possible.…”

Section: Substrate Bindingmentioning

confidence: 99%

“…In subtilisin and thermitase, the specificity appears to be largely determined by interactions of the substrate P4 and PI residue side chains with the large, hydrophobic S4 and S I subsites (Gron et al, 1992). In NisP, the predicted subsites can be described as follows (summary in Table II): S4, a distinct pocket with the hydrophobic residues F140, VI50 and A 169 at one side and the more hydrophilic residues S145, S147, Q171 and YI92 (hydrogen bonded to each other) at the other side; S3, not a distinct site, since the substrate P3 residue points away from the enzyme towards the solvent; S2, a shallow pocket, bounded at the sides and bottom by FI40 and active site residue Hill, and at the rim by residue N143; SI, a large, elongated pocket with residue D241 at the bottom and D213 at the rim.…”

Section: Substrate Bindingmentioning

confidence: 99%

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Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

Siezen¹,

Rollema²,

Kuipers³

et al. 1995

Protein Eng Des Sel

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Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 12-05-2018Protein Engineering vol.8 no.2 pp. [117][118][119][120][121][122][123][124][125] 1995 Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin ITo whom correspondence should be addressed A model is presented for the 3-D structure of the catalytic domain of the putative leader peptidase NisP of Lactococcus lactis, and the interaction with its specific substrate, the precursor of the lantibiotic nisin. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin. Predictions are made of the general protein fold, inserted loops, Ca2+ binding sites, aromatic interactions and electrostatic interactions of NisP. Cleavage of the leader peptide from precursor nisin by NisP is the last step in maturation of nisin. A detailed prediction of the substrate binding site attempts to explain the basis of specificity of NisP for precursor nisin. Specific acidic residues in the SI sub site of the substrate binding region of NisP appear to be of particular importance for electrostatic interaction with the PI Arg residue of precursor nisin after which cleavage occurs. The hydrophobic S4 subsite of NisP may also contribute to substrate binding as it does in subtilisins. Predictions of enzyme-substrate interaction were tested by protein engineering of precursor nisin and determining susceptibility of mutant precursors to cleavage by NisP. An unusual property of NisP predicted from this catalytic domain model is a surface patch near the substrate binding region which is extremely rich in aromatic residues. It may be involved in binding to the cell membrane or to hydrophobic membrane proteins, or it may serve as the recognition and binding region for the modified, hydrophobic C-terminal segment of precursor nisin. Similar predictions for the tertiary structure and substrate binding are made for the highly homologous protein EpiP, the putative leader peptidase for the lantibiotic epidermin from Staphylococcus epidermidis, but EpiP lacks the aromatic patch. Based on these models, protein engineering can be employed not only to test the predicted enzyme-substrate interactions, but also to design lantibiotic leader peptidases with a desired specificity.

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Section: Discussionmentioning

confidence: 77%

Section: Substrate Bindingmentioning

confidence: 99%

Section: Substrate Bindingmentioning

confidence: 99%

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Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

Siezen¹,

Rollema²,

Kuipers³

et al. 1995

Protein Eng Des Sel

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“…The linkages between protein domains are often particularly susceptible to proteolysis. Subtilisin is known as a protease having a broad subsite specificity [36,37]. Therefore, it is comprehensible that proteins possibly are readily digested when employed in subtilisin treatment.…”

Section: Discussionmentioning

confidence: 99%

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

Stoller¹,

Tradler²,

Rücknagel³

et al. 1996

FEBS Letters

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The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl-prolyl cisltrans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatieally active fragment could be isolated after proteolysis by subtifisin. The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251. The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His-tagged polypeptide was isolated on an Ni 2+-NTA column. Subsequent digestion with subtifisin and anionexchange chromatography yielded a TF fragment encompassing amino acids Gin-148 to Thr-249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 ~1-1 s -l could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrofide FK506.

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“…G T ‡ (changes in transition state stabilization energy) of the peptides was calculated using the relative difference in k cat /K m values [28], where G T ‡ is negative if the X-X-X-Arg peptide is a better substrate than the Y-Y-Y-Arg peptide (where X and Y are any amino acid):…”

Section: Figure 1 Specificity Profile Of Rfviia In the Presence Of Stfmentioning

confidence: 99%

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

Larsen

Østergaard

Bjelke

et al. 2007

Biochemical Journal

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The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. In the present study, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning substrate combinatorial libraries and evaluated the role of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen. To evaluate the role of the active-site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (position 99 in chymotrypsin), which is considered to be one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.

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Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching

Cited by 142 publications

References 31 publications

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

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