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PDBsum entry 3cms

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protein links
Hydrolase PDB id
3cms
Jmol
Contents
Protein chain
320 a.a. *
Waters ×145
* Residue conservation analysis
PDB id:
3cms
Name: Hydrolase
Title: Engineering enzyme sub-site specificity: preparation, kineti characterization and x-ray analysis at 2.0-angstroms resolu val111phe site-mutated calf chymosin
Structure: Chymosin b. Chain: a. Engineered: yes
Source: Bos taurus. Cattle. Organism_taxid: 9913
Resolution:
2.00Å     R-factor:   0.195    
Authors: M.Newman,C.Frazao,A.Shearer,I.J.Tickle,T.L.Blundell
Key ref:
P.Strop et al. (1990). Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin. Biochemistry, 29, 9863-9871. PubMed id: 2271625 DOI: 10.1021/bi00494a016
Date:
26-Feb-90     Release date:   15-Oct-92    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P00794  (CHYM_BOVIN) -  Chymosin
Seq:
Struc:
381 a.a.
320 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.3.4.23.4  - Chymosin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Broad specificity similar to that of pepsin A. Clots milk by cleavage of a single bond in casein (kappa chain).
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     proteolysis   1 term 
  Biochemical function     aspartic-type endopeptidase activity     1 term  

 

 
DOI no: 10.1021/bi00494a016 Biochemistry 29:9863-9871 (1990)
PubMed id: 2271625  
 
 
Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin.
P.Strop, J.Sedlacek, J.Stys, Z.Kaderabkova, I.Blaha, L.Pavlickova, J.Pohl, M.Fabry, V.Kostka, M.Newman.
 
  ABSTRACT  
 
Comparison of the three-dimensional structure of bovine chymosin with the structures of homologous aspartic proteinases complexed with peptide inhibitors shows that Val111 in chymosin occupies a position between the specificity subsites S1 and S3. A mutation corresponding to Val111 to Phe has been introduced in an intermediary plasmid construct of prochymosin by bridging its unique restriction sites by a synthetic mutant oligonucleotide duplex. A prochymosin fusion product was expressed in Escherichia coli in such a way that the extension and substitution of the propart does not interfere with the activation of the zymogen. After activation of the crude prochymosin, the enzyme was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as ligand. This procedure provided large amounts of pure protein as judged by FPLC, the activity/protein ratio, and SDS-PAGE. The enzymatic properties were determined by using a variety of peptide substrates and inhibitors; KM values for the mutant enzyme were approximately twice those of the wild type, but the kcat values were little changed. The mutant enzyme was crystallized, X-ray data were collected to 2.0-A resolution by using a FAST area detector, and the structure was solved by using difference Fourier methods and refined to an R factor of 19.5%. The mutation leads to only local changes in conformation, with the phenylalanine side chain occupying part of the S1 and S3 pockets. This accounts for the increased KM of this mutant for a substrate with a large phenylalanine side chain at P1. It is also consistent with the higher affinity of the mutant for an inhibitor with small side chains at P1 and P3 when compared with the wild-type enzyme.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
17447722 C.L.Parr, R.A.Keates, B.C.Bryksa, M.Ogawa, and R.Y.Yada (2007).
The structure and function of Saccharomyces cerevisiae proteinase A.
  Yeast, 24, 467-480.  
16216580 A.A.Gorfe, and A.Caflisch (2005).
Functional plasticity in the substrate binding site of beta-secretase.
  Structure, 13, 1487-1498.  
15039581 M.O.Badasso, V.Dhanaraj, S.P.Wood, J.B.Cooper, and T.L.Blundell (2004).
Crystallization and X-ray analysis of the Y75N mutant of Mucor pusillus pepsin complexed with inhibitor.
  Acta Crystallogr D Biol Crystallogr, 60, 770-772.  
11714911 N.S.Andreeva, and L.D.Rumsh (2001).
Analysis of crystal structures of aspartic proteinases: on the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes.
  Protein Sci, 10, 2439-2450.  
18636608 S.C.Burton, N.W.Haggarty, and D.R.Harding (1997).
One step purification of chymosin by mixed mode chromatography.
  Biotechnol Bioeng, 56, 45-55.  
9228062 T.Shintani, K.Nomura, and E.Ichishima (1997).
Engineering of porcine pepsin. Alteration of S1 substrate specificity of pepsin to those of fungal aspartic proteinases by site-directed mutagenesis.
  J Biol Chem, 272, 18855-18861.  
8673731 G.Houen, M.T.Madsen, K.W.Harlow, P.Lønblad, and B.Foltmann (1996).
The primary structure and enzymic properties of porcine prochymosin and chymosin.
  Int J Biochem Cell Biol, 28, 667-675.  
1518925 D.Shortle (1992).
Mutational studies of protein structures and their stabilities.
  Q Rev Biophys, 25, 205-250.  
1367681 A.C.Storer (1991).
Engineering of proteases and protease inhibition.
  Curr Opin Biotechnol, 2, 606-613.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time.