Chemical modification of 3.alpha.,20.beta.-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue

Pasta, Piero; Mazzola, G.; Carrea, Giacomo

doi:10.1021/bi00379a007

Cited by 24 publications

(13 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Presumably, the ~4C incorporations now first obtained corresponded to low amounts of Cys-containing contaminating protein chains, or to alkylation non-specific for Cys residues. Other reports mentioning Cys [16,17] also seem to lack specific proof. Combined, the different complications may indicate a reduced reliability at single positions.…”

Section: Directmentioning

confidence: 93%

“…Finally, lack of Cys in the amino acid sequence (Fig. 2) was unexpected since a Cys residue has been postulated close to the steroid binding site [16,17] and a ~4C incorporation corresponding to 0.2-1 mol/mol was now initially found upon iodoacetate treatment, but could not be detected in the peptides, nor in the preparation analyzed (<0.3 mol/mol). Presumably, the ~4C incorporations now first obtained corresponded to low amounts of Cys-containing contaminating protein chains, or to alkylation non-specific for Cys residues.…”

Section: Directmentioning

confidence: 99%

See 1 more Smart Citation

Prokaryotic 20β‐hydroxysteroid dehydrogenase is an enzyme of the ‘short‐chain, non‐metalloenzyme’ alcohol dehydrogenase type

1990

View full text Add to dashboard Cite

The primary structure of 20fl-hydroxysteroid dehydrogenase from Streptomyces hydrogenans was determined after FPLC purification of a commercial preparation. Peptides obtained from different proteolytic cleavages were purified by reverse phase HPLC. The 255-residue structure deduced was found to be distantly homologous to those of Drosophila alcohol dehydrogenase and several other dehydrogenases, establishing that prokaryotic 20fl-hydroxysteroid dehydrogenase as a member of the 'short-chain alcohol dehydrogenase family'. With the enzymes characterized, the identity is greatest (31-34%) towards 4 other prokaryotic dehydrogenases, but the family also includes mammalian steroid and prostaglandin dehydrogenases. These enzymes are low in Cys and have a strictly conserved Tyr residue that appears to be important.

show abstract

Section: Directmentioning

confidence: 93%

Section: Directmentioning

confidence: 99%

Prokaryotic 20β‐hydroxysteroid dehydrogenase is an enzyme of the ‘short‐chain, non‐metalloenzyme’ alcohol dehydrogenase type

1990

View full text Add to dashboard Cite

show abstract

“…However, the His residue claimed to be "affinity alkylated" by haloacetoxysteroids and chemically modified by diethyl pyrocarbonate (ref. 28 and references therein) is His-181, which is buried enough (Fig. 2C) to be inaccessible without some rearrangements of the active-site structure in this holo form.…”

Section: This Puts the Nad(h) Binding Region (3a-ab-(b--ac-(3c-mentioning

confidence: 96%

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

Ghosh

Weeks²,

Grochulski³

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

224

188

View full text Add to dashboard Cite

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3a,20/3-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the a/( fold characteristic ofthe dinucleotide binding region. The fold of the rest of the subunit, the quarternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3a-and 2013-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. (1), which includes 11f,-hydroxysteroid (llP-HSD) (2), 7a-hydroxysteroid (3), and 15-hydroxyprostaglandin dehydrogenases (4) from mammals; glucose (5) and ribitol (6) dehydrogenases, as well as a putative nodulation factor (7) from bacteria; and an ADH (8) from insects. Enzymes belonging to this family have -250 amino acid residues, similar coenzyme specificity, and partial sequence homology. Although more than 40 crystal structures of =15 types of NAD(H)-and NADP(H)-linked dehydrogenase enzymes have been determined at medium-to-high resolution (9), to our knowledge no x-ray crystallographic study describing the three-dimensional structure of a dehydrogenase belonging to this short-chain class has been reported. This is only the third structure of an enzyme for which steroids are the substrate to be determined by x-ray diffraction techniques. A lowresolution structure of keto-steroid isomerase (10) and the refined structure of cholesterol oxidase (11) have been published.To account for the ability of 3a,20f3-HSD to transfer a hydride to either end of a steroid molecule, "one steroid-two cofactor sites" and "two steroid orientations-one cofactor site" models (12) have been proposed. When analyzed in conjunction with sequence homology studies, the threedimensional structured especially at the cofactor binding and the substrate binding regions, offers further insight concerning the significance of conserved residues and their possible roles in substrate specificity and overall enzyme function. MATERIALS AND METHODSThe crystals, grown in the presence of 4 mM NADH, belong to the space group P43212 having unit cell dimensions a = 106.2 A and c = 203.8 A and contain one full tetramer (106 kDa) in the asymmetric unit (13 .091], was collected on film from six crystals at the Cornell High Energy Synchrotron Source and processed by using Rossmann's program at Purdue University. The area detector data to 3 A and film data between 3 and 2.6 A were merged into a composite native data set. The Hg derivatives each had a single major binding site per subunit, whereas the Au reagent gave a "multip...

show abstract

“…This indicates that hydroxylamine reverses the histidine modification by DEPC and strongly suggests that it reverses the lysine modification as well. DEPC modification of highly reactive lysine residues in proteins can be reversed by hydroxylamine as reported by Pasta et al (30), although an earlier report had concluded that reversibility was specific for histidine (16). Thus, reversal of DEPC modification of both histidine and lysine residues in the heparin-binding site of selenoprotein P seems likely because both histidine and lysine residues clearly contribute to the protein-heparin interaction.…”

Section: Discussionmentioning

confidence: 78%

“…Details of the experiment are given under "Experimental Procedures." hydroxylamine treatment can also reverse modification of lysine residues (30). This should be especially true in the case of highly reactive lysine residues.…”

Section: Table IV Mass Mapping Analysis Of Peptides Bound To Heparinmentioning

confidence: 99%

Heparin-binding Histidine and Lysine Residues of Rat Selenoprotein P

Hondal¹,

Caprioli

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Selenoprotein P is a plasma protein that has oxidant defense properties. It binds to heparin at pH 7.0, but most of it becomes unbound as the pH is raised to 8.5. This unusual heparin binding behavior was investigated by chemical modification of the basic amino acids of the protein. Diethylpyrocarbonate (DEPC) treatment of the protein abolished its binding to heparin. DEPC and [ 14 C]DEPC modification, coupled with amino acid sequencing and matrix-assisted laser desorption ionizationtime of flight mass spectrometry of peptides, identified several peptides in which histidine and lysine residues had been modified by DEPC. Two peptides from one region (residues 80 -95) were identified by both methods. Moreover, the two peptides that constituted this sequence bound to heparin. Finally, when DEPC modification of the protein was carried out in the presence of heparin, these two peptides did not become modified by DEPC. Based on these results, the heparin-binding region of the protein sequence was identified as KHAHLKKQVS-DHIAVY. Two other peptides (residues 178-189 and 194-234) that contain histidine-rich sequences met some but not all of the criteria of heparin-binding sites, and it is possible that they and the histidine-rich sequence between them bind to heparin under some conditions. The present results indicate that histidine is a constituent of the heparin-binding site of selenoprotein P. The presence of histidine, the pK a of which is 7.0, explains the release of selenoprotein P from heparin binding as pH rises above 7.0. It can be speculated that this property would lead to increased binding of selenoprotein P in tissue regions that have low pH.

show abstract

Chemical modification of 3.alpha.,20.beta.-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue

Cited by 24 publications

References 24 publications

Prokaryotic 20β‐hydroxysteroid dehydrogenase is an enzyme of the ‘short‐chain, non‐metalloenzyme’ alcohol dehydrogenase type

Prokaryotic 20β‐hydroxysteroid dehydrogenase is an enzyme of the ‘short‐chain, non‐metalloenzyme’ alcohol dehydrogenase type

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

Heparin-binding Histidine and Lysine Residues of Rat Selenoprotein P

Contact Info

Product

Resources

About