Steroid Delta-isomerase

steroid delta-isomerase
Identifiers
EC number 5.3.3.1
CAS number 9031-36-1
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO

In enzymology, a steroid Delta-isomerase (EC 5.3.3.1) is an enzyme that catalyzes the chemical reaction

a 3-oxo-Delta5-steroid \rightleftharpoons a 3-oxo-Delta4-steroid

Hence, this enzyme has one substrate, 3-oxo-Delta5-steroid, and one product, 3-oxo-Delta4-steroid.

Contents

Introduction

This enzyme belongs to the family of isomerases, specifically those intramolecular oxidoreductases transposing C=C bonds. The systematic name of this enzyme class is 3-oxosteroid Delta5-Delta4-isomerase. Other names in common use include ketosteroid isomerase (KSI), hydroxysteroid isomerase, steroid isomerase, Delta5-ketosteroid isomerase, Delta5(or Delta4)-3-keto steroid isomerase, Delta5-steroid isomerase, 3-oxosteroid isomerase, Delta5-3-keto steroid isomerase, and Delta5-3-oxosteroid isomerase.

KSI has been studied extensively from the bacteria Comamonas testosteroni (TI), formerly referred to as Pseudomonas testosteroni, and Pseudomonas putida (PI).[1] Mammalian KSI has been studied from bovine adrenal cortex[2] and rat liver.[3] This enzyme participates in c21-steroid hormone metabolism and androgen and estrogen metabolism. An example substrate is delta-5-androstene-3,17-dione, which KSI converts to delta-4-androstene-3,17-dione.[4] The above reaction in the absence of enzyme takes 7 weeks to complete in aqueous solution.[5] KSI performs this reaction on an order of 1011 times faster, ranking it among the most proficient enzymes known [5]. Bacterial KSI also serves as a model protein for studying enzyme catalysis[6] and protein folding.[7]

Structural studies

KSI exists as a homodimer with two identical halves.[7] The interface between the two monomers is narrow and well defined, consisting of neutral or apolar amino acids, suggesting the hydrophobic interaction is important for dimerization.[8] Results show that the dimerization is essential to function.[7] The active site is highly apolar and folds around the substrate in a manner similar to other enzymes with hydrophobic substrates, suggesting this fold is characteristic for binding hydrophobic substrates.[9]

As of late 2007, 25 structures have been solved for this class of enzymes, with PDB accession codes 1BUQ, 1C7H, 1CQS, 1DMM, 1DMN, 1DMQ, 1E97, 1GS3, 1ISK, 1K41, 1OCV, 1OGX, 1OGZ, 1OH0, 1OHO, 1OHP, 1OHS, 1OPY, 1VZZ, 1W00, 1W01, 1W02, 1W6Y, 2PZV, and 8CHO.

Mechanism

The conversion of a Delta-5 steroid to a conjugated system Delta-4 steroid begins with Asp-38 abstracting a hydrogen at the 4 position to form an enolate.[1] Asp-38 then transfers the proton proton to the 6 position to give the product [1]

There have been conflicting results on the ionization state of the intermediate, whether it exists as the enolate[10] or enol.[11] Pollack uses a thermodynamic argument to suggest the intermediate exists as the enolate.[1]

Also of contention is the nature of the hydrogen bonding network stabilizing the reaction intermediate, whether Tyr-14 and Asp-99 both form hydrogen bonds directly to the O-3[12] or Asp-99 hydrogen bonds to Tyr-14 which hydrogen bonds to O-3.[13]

Biological Function

KSI occurs in animal tissues concerned with steroid hormone biosynthesis, such as the adrenal, testis, and ovary.[14] KSI in Comamomas testosteroni is used in the degradation pathway of steroids, allowing this bacteria to utilize steroids containing a double bond at delta-5, such as testosterone, as its sole source of carbon.[15]

Model Enzyme

KSI has been used as a model system to test different theories to explain how enzymes achieve their catalytic efficiency. Low-barrier hydrogen bonds and unusual pKa values for the catalytic residues have been proposed as the basis for the fast action of KSI.[9] Another proposal explaining enzyme catalysis tested through KSI is the geometrical complementarity of the active site to the transition state, which proposes the active site electrostatics is complementary to the substrate transition state.[6]

KSI has also been a model system for studying protein folding. Kim et al. studied the effect of folding and tertiary structure on the function of KSI.[7]

References

  1. ^ a b c d Pollack, R (2004). "Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase". Bioorganic Chemistry 32 (5): 341–53. doi:10.1016/j.bioorg.2004.06.005. PMID 15381400. 
  2. ^ Bertolino, A; Benson, A; Talalay, P (1979). "Activation of Δ5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins". Biochemical and Biophysical Research Communications 88 (3): 1158–66. doi:10.1016/0006-291X(79)91530-4. PMID 465075. 
  3. ^ Benson, A; Talalay, P (1976). "Role of reduced glutathione in the Δ5-3-ketosteroid isomerase reaction of liver". Biochemical and Biophysical Research Communications 69 (4): 1073–9. doi:10.1016/0006-291X(76)90482-4. PMID 6023. 
  4. ^ Talalay, Paul; Benson, Ann M (1972). "Δ5-3-Ketosteroid Isomerase". In Boyer, Paul D.. The Enzymes. 6 (3rd ed.). Academic Press. pp. 591–618. ISBN 978-0-12-122706-7. http://books.google.com/books?id=_YP9Qu299UwC&pg=PA591. 
  5. ^ a b Radzicka, A; Wolfenden, R (1995). "A proficient enzyme". Science 267 (5194): 90–3. doi:10.1126/science.7809611. PMID 7809611. 
  6. ^ a b Kraut, Daniel A.; Sigala, Paul A.; Pybus, Brandon; Liu, Corey W.; Ringe, Dagmar; Petsko, Gregory A.; Herschlag, Daniel (2006). "Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole". PLoS Biology 4 (4): e99. doi:10.1371/journal.pbio.0040099. PMC 1413570. PMID 16602823. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1413570. 
  7. ^ a b c d Kim, D.-H.; Nam, GH; Jang, DS; Yun, S; Choi, G; Lee, HC; Choi, KY (2001). "Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B". Protein Science 10 (4): 741–52. doi:10.1110/ps.18501. PMC 2373975. PMID 11274465. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2373975. 
  8. ^ Kim, D.-H.; Nam, GH; Jang, DS; Yun, S; Choi, G; Lee, HC; Choi, KY (2001). "Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B". Protein Science 10 (4): 741–52. doi:10.1110/ps.18501. PMC 2373975. PMID 11274465. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2373975.  cited in Kim, D.-H.; Nam, GH; Jang, DS; Yun, S; Choi, G; Lee, HC; Choi, KY (2001). "Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B". Protein Science 10 (4): 741–52. doi:10.1110/ps.18501. PMC 2373975. PMID 11274465. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2373975. 
  9. ^ a b Ha, N.-C.; Kim, MS; Lee, W; Choi, KY; Oh, BH (2000). "Detection of Large pKa Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes". Journal of Biological Chemistry 275 (52): 41100–6. doi:10.1074/jbc.M007561200. PMID 11007792. 
  10. ^ Xue, Liang; Kuliopulos, Athan; Mildvan, Albert S.; Talalay, Paul (1991). "Catalytic mechanism of an active-site mutant (D38N) of .DELTA.5-3-ketosteroid isomerase". Biochemistry 30 (20): 4991–7. doi:10.1021/bi00234a022. PMID 2036366. 
  11. ^ Petrounia, Ioanna P.; Pollack, Ralph M. (1998). "Substituent Effects on the Binding of Phenols to the D38N Mutant of 3-Oxo-Δ5-steroid Isomerase. A Probe for the Nature of Hydrogen Bonding to the Intermediate". Biochemistry 37 (2): 700–5. doi:10.1021/bi972262s. PMID 9425094. 
  12. ^ Wu, Z. R.; Ebrahimian, S; Zawrotny, ME; Thornburg, LD; Perez-Alvarado, GC; Brothers, P; Pollack, RM; Summers, MF (1997). "Solution Structure of 3-Oxo-5-Steroid Isomerase". Science 276 (5311): 415–8. doi:10.1126/science.276.5311.415. PMID 9103200. 
  13. ^ Zhao, Qinjian; Abeygunawardana, Chitrananda; Gittis, Apostolos G.; Mildvan, Albert S. (1997). "Hydrogen Bonding at the Active Site of Δ5-3-Ketosteroid Isomerase†". Biochemistry 36 (48): 14616–26. doi:10.1021/bi971549m. PMID 9398180. 
  14. ^ Kawahara, Frank S.; Wang, Shu-Fang; Talalay, Paul (1962). "The Preparation and Properties of Crystalline Δ5-3-Ketosteroid Isomerase". The Journal of Biological Chemistry 237: 1500–6. PMID 14454546. http://www.jbc.org/cgi/pmidlookup?view=long&pmid=14454546. 
  15. ^ Talalay, Paul; Dobson, Marie Mollomo; Tapley, Donald F. (1952). "Oxidative Degradation of Testosterone by Adaptive Enzymes". Nature 170 (4328): 620–1. doi:10.1038/170620a0. PMID 13002385. 

Further reading

Isomerases: intramolecular oxidoreductases (EC 5.3)
5.3.1: Aldoses/Ketoses
5.3.2: Keto/Enol
5.3.3: C=C
5.3.4: S-S
5.3.99: other
B enzm: 1.1/2/3/4/5/6/7/8/10/11/13/14/15-18, 2.1/2/3/4/5/6/7/8, 2.7.10, 2.7.11-12, 3.1/2/3/4/5/6/7, 3.1.3.48, 3.4.21/22/23/24, 4.1/2/3/4/5/6, 5.1/2/3/4/99, 6.1-3/4/5-6