Affinity alkylation of the active site of C/sub 21/ steroid side-chain cleavage cytochrome P-450 from neonatal porcine testis: a unique cysteine residue alkylated by 17-(bromoacetoxy)progesterone
The affinity alkylating progesterone analogue 17-(bromoacetoxy)progesterone has been used to label the active site of a microsomal cytochrome P-450 enzyme from neonatal pig testis. The enzyme causes removal of the C/sub 20/ and C/sub 21/ side chains from the substrates progesterone and pregnenolone by catalyzing both 17-hydroxylase and C/sub 17,20/-lyase reactions, which produce the corresponding C/sub 1//sup 9/ steroidal precursors of testosterone. The progesterone analogue causes simultaneous inactivation of the two catalytic activities of the enzyme by a first-order kinetic process that obeys saturation kinetics. Progesterone and 17-hydroxyprogesterone each protect the enzyme against inactivation. The progesterone analogue is a competitive inhibitor of the enzyme with K/sub i/ values of 8.4 ..mu..M and 7.8 ..mu..M for progesterone and 17-hydroxyprogesterone, respectively. The enzyme inactivation and kinetic data are consistent with a theory proposing that the analogue and the two substrates compete for the same active site. The radioactive analogue 17-((/sup 14/C)bromoacetoxy)progesterone causes inactivation of the enzyme with incorporation of 1.5-2.2 mol of the analogue per mole of inactivated enzyme. When this experiment is carried out in the presence of a substrate, then 0.9-1.2 mol of radioactive analogue is incorporated per mole of inactivated enzyme. The data suggest that the analogue can bind to two different sites, one of which is related to the catalytic site. Radiolabeled enzyme samples, from reactions of the /sup 14/C-labeled analogue with the enzyme alone or with enzyme in the presence of a substrate, were subjected to amino acid analysis and also in tryptic digestion and peptide mapping.
- Research Organization:
- Worcester Foundation for Experimental Biology, Shrewburg, MA
- OSTI ID:
- 6620045
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:2; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ALKYLATION
AMINO ACID SEQUENCE
AMINO ACIDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CYSTEINE
CYTOCHROMES
DECOMPOSITION
DOMESTIC ANIMALS
ENZYMATIC HYDROLYSIS
ENZYMES
GONADS
HORMONES
HYDROLYSIS
KETONES
KINETICS
LABELLED COMPOUNDS
LYSIS
MALE GENITALS
MAMMALS
MOLECULAR STRUCTURE
NEONATES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PIGMENTS
PREGNANES
PROGESTERONE
PROTEINS
REACTION KINETICS
SOLVOLYSIS
STEROID HORMONES
STEROIDS
SWINE
TESTES
THIOLS
VERTEBRATES