Synthetic active site analogues of heme-thiolate proteins. Characterization and identification of intermediates of the catalytic cycles of cytochrome P450cam and chloroperoxidase.

In view of recent results from different sources, the reaction mechanisms of two heme-thiolate proteins, cytochrome P450cam and chloroperoxidase (CPO), are discussed. In this context a mechanism of CPO is proposed which includes H2O2 cleavage, subsequent formation of compound I and the identification of two elusive intermediates. The HOCl adduct of the iron(III)porpyhrin is the catalytically competent Cl+ donor chlorinating activated C-H bonds of substrates bound to the enzyme. Pulse-EPR characterization of an enzyme model of the resting state of P450cam suggests a role of the electric field of the protein for stabilizing the low-spin state of the cofactor of the enzyme. It is further suggested that the same effect of the protein may trigger the reactivity of compound I such that both concerted and two-step reactions are feasible within the concept of a Two-State-Reactivity. This review emphasizes the value of synthetic enzyme models complementing investigations of the native proteins.

Expression pattern and localization of beta,beta-carotene 15,15'-dioxygenase in different tissues.

Beta,beta-carotene 15,15'-dioxygenase cleaves beta,beta-carotene into two molecules of retinal, and is the key enzyme in the metabolism of beta,beta-carotene to vitamin A. The enzyme has been known for more than 40 years, yet all attempts to purify the protein to homogeneity have failed. Recently, the successful cloning and sequencing of an enzyme with beta,beta-carotene 15,15'-dioxygenase activity from chicken, as well as from Drosophila, has been reported. Here, we describe in detail our attempt to enrich the chicken beta,beta-carotene 15,15'-dioxygenase to such an extent as to allow determination of partial amino acid sequences, which were then used to design degenerate oligonucleotides. Screening of a chicken duodenal expression library yielded a full-length clone containing a coding sequence of 1578 bp. Functional expression in Escherichia coli and in eukaryotic cell lines confirmed that we had cloned the first vertebrate dioxygenase that cleaves beta,beta-carotene at the central 15,15'-double bond. By performing a sequence homology search, the cDNA sequence of the mouse homologue was found as an expressed sequence tag (EST) in the gene bank. At the amino-acid level, the degree of homology between the chicken and mouse sequences is 81%. Thus beta,beta-carotene 15,15'-dioxygenase can be considered as being an enzyme that is evolutionarily rather well conserved. We established the expression pattern of beta,beta-carotene 15,15'-dioxygenase in chicken and mouse tissues with a combination of Northern blots and in situ hybridization. The mRNA for beta,beta-carotene 15,15'-dioxygenase was localized primarily in duodenal villi, as well as in liver and in tubular structures of lung and kidney. These new findings demonstrate that beta,beta-carotene 15,15'-dioxygenase is also expressed in epithelial structures, where it serves to provide the tissue-specific vitamin A supply.

Cloning and expression of beta,beta-carotene 15,15'-dioxygenase.

beta,beta-Carotene 15,15'-dioxygenase cleaves beta-carotene into two molecules of retinal and is therefore the key enzyme in beta-carotene metabolism to vitamin A. In the present study, it was possible to enrich the chicken beta,beta-carotene 15,15'-dioxygenase to such an extent that partial amino acid sequence information could be obtained to design degenerate oligonucleotides. With RT-PCR a cDNA fragment could be obtained and used subsequently in a radioactive screening of a chicken duodenal expression library. We cloned the first eukaryotic beta,beta-carotene 15,15'-dioxygenase which symmetrically cleaves beta-carotene at the 15,15'-double bond.

Identification of intermediates in the catalytic cycle of chloroperoxidase.

BACKGROUND: Chloroperoxidase (CPO) is the most versatile of the hemethiolate proteins, catalyzing the chlorination of activated C-H bonds and reactions reminiscent of peroxidase, catalase, and cytochrome P450. Despite 30 years of continuous efforts, no intermediates of the enzyme's catalytic cycle have been identified except for compound I. Thus, in the absence of conclusive evidence it is generally believed that the halogenation of substrates proceeds by means of 'free HOCI' in solution. RESULTS: The pH profile of chloroperoxidase from Caldariomyces fumago revealed a new active-site complex that can be detected only at pH 4.4. According to ultra-violet (UV) spectroscopy, and by comparison with suitable enzyme models, this intermediate is the HOCl adduct of the iron(III) protoporphyrin(IX). Inactivation of chloroperoxidase by diethyl pyrocarbonate, which interrupts the proton shuttle by modification of the distal histidine, led to the formation of the -OCl adduct of the iron complex, which was identified by comparison with a corresponding active site analogue. CONCLUSIONS: The availability of enzyme models of heme-thiolate proteins allowed the identification by UV spectroscopy of both the -OCl adduct and the HOCl adduct of the iron(III) protoporphyrin(IX) of chloroperoxidase. The existence of these previously elusive intermediates suggests that the chlorination catalyzed by CPO, and its corresponding active site analogue, proceeds by Cl+ transfer from the HOCl adduct to the substrate bound in the distal pocket of the enzyme.

The substrate specificity of tocopherol cyclase.

The substrate specificity of the enzyme tocopherol cyclase from the blue-green algae Anabaena variabilis (Cyanobacteria) was investigated with 11 substrate analogues revealing the significance of three major recognition sites: (i) the OH group at C(1) of the hydroquinone, (ii) the (E) configuration of the double bond, and (iii) the length of the lipophilic side chain. Experiments with two affinity matrices suggest that substrates approach the enzyme's active site with the hydrophobic tail.