Ann M. Benson

The amino acid sequence of Clostridium butyricum ferredoxin

Abstract The amino acid sequence of Clostridium butyricum ferredoxin, determined by the application of conventional methods of sequence determination primarily to the S -β-aminoethyl protein, was found to be: Ala-Phe-Val-Ile-Asn-Asp-Ser-Cys-Val-Ser- Cys-Gly-Ala-Cys-Ala-Gly-Glu-Cys-Pro-Val-Ser-Ala-Ile-Thr-Gln-Gly-Asp-Thr-Gln- Phe-Val-Ile-Asp-Ala-Asp-Thr-Cys-Ile-Asp-Cys-Gly-Asn-Cys-Ala-Asn-Val-Cys-Pro- Val-Gly-Ala-Pro-Asn-Gln-Glu. This sequence differs in nine positions from that of Clostridium pasteurianum ferredoxin. The positions of the cysteine and proline residues are invariant in these two species, as is the sequence including residues 30–52.

The amino acid sequence of bovine heart cytochrome c

Abstract The amino acid composition of beef heart cytochrome c was determined. The protein was digested with chymotrypsin and trypsin and the digests were resolved by ion exchange chromatography on Dowex 50 with volatile buffers. From the chymotryptic and tryptic digests, 16 and 17 fractions, respectively, were isolated and characterized. From the overlapping composition and sequences plus the use of the chemical reagents, cyanogen bromide and N-bromosuccinimide, the complete sequence of 104 residues was deduced. Bovine heart cytochrome c has an amino acid sequence identical with that of pig and sheep cytochromes c.

[71] Steroid-transforming enzymes

Publisher Summary Affinity chromatography on adsorbents containing steroidal substrate analogs covalently linked to agarose promises to be an extremely valuable technique both for the purification of steroid-transforming enzymes, and for studies on their mechanism. When Pseudomonas testosteroni is grown on media containing certain steroids, crude extracts of lyophilized or sonically disrupted cells are a rich source of various induced steroid transforming enzymes. The isolation from these mixtures of discrete catalytic proteins, in purified form and uncontaminated by each other, has been a challenging task. The availability of such purified enzymes would greatly augment their suitability for the microanalysis and determination of steric configuration of steroids. Affinity techniques have indicated that the fl-enzymes do not bind strongly to steroids in the absence of NAD + , thus suggesting that these oxidations may involve an obligatory ordered addition in which the binding of NAD + precedes that of the steroidal substrate.

18 Δ5-3-Ketosteroid Isomerase

Publisher Summary This chapter discusses the molecular and catalytic properties of ∆ 5 -3-ketosteroid isomerase. The ∆ 5 -3-ketosteroid isomerase promotes the conversion of a variety of ∆ 5(6) - and ∆ 5(10) -3-ketosteroids to the corresponding ∆ 4 -3-ketosteroids. Although the transformation of a variety of ∆ 5 -3-hydroxysteroids to ∆ 4 -3-ketosteroids had been described in a number of crude enzymic systems of animal, vegetable, and bacterial origin, it had not been appreciated that these conversions involved two enzymic steps, including a freely reversible nicotinamide-adenine nucleotide-dependent oxidation of the hydroxyl group to the ketone, followed by a largely irreversible transposition of the double bond into a position of conjugation. The ultraviolet absorption spectrum of crystalline isomerase in dilute sodium phosphate buffer at pH 7.0 shows a principal absorption peak at 277 nm and a well-defined shoulder at 282–284 nm, both of which are characteristic of tyrosine, although slightly displaced toward longer wavelengths than in the case of a free amino acid. In addition, the intact protein displays clearly defined maxima at 253, 259, 266, and 269 nm, which are diagnostic of phenylalanine absorptions but are also displaced toward longer wavelengths.

[51] Irreversible inhibitors of Δ5-3-ketosteroid isomerase: Acetylenic and allenic 3-oxo-5,10-secosteroids

Publisher Summary The Δ-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosterone catalyzes the conversion of a variety of unconiugated Δ 5(6)- and Δ -3-ketosteroids into the corresponding Δ-3-ketosteroids. Typical examples of the reaction are the conversion of Δ-androstene-3,17-dione and Δ-pregnene-3,20-dione to the respective Δ -3-ketosteroids. Considerable information is available on the molecular properties of this enzyme, on the catalytic mechanism, and on the stereochemistry of the enzymic reaction. Based on the proposed molecular mechanism of this reaction, a series of acetylenic 5,10-secosteroids has been prepared in the belief that they might serve as substrates for Δ 5-3-ketosteroid isomerase. Abstraction of the proton at C-4 by the enzyme should then generate, via an enolic intermediate, the corresponding highly reactive conjugated allenic ketones, which might be expected to react covalently with a nucleophilie amino acid residue at the active site. This chapter is based on expected conformational similarities between the acetylenie 5,10-secosteroids and the normal Δ-3-ketosteroid substrates for the enzyme.