Carbonyl reduction plays a significant role in physiological processes throughout the body. Although much is known about endogenous carbonyl metabolism, much less is known about the roles of carbonyl-reducing enzymes in xenobiotic metabolism. Multiple pathways exist in humans for metabolizing carbonyl moieties of xenobiotics to their corresponding alcohols, readying these molecules for subsequent conjugation and/or excretion. When exploring carbonyl reduction clearance pathways for a drug development candidate, it is possible to assess the relative contributions of these enzymes due to their differences in subcellular locations, cofactor dependence, and inhibitor profiles. In addition, the contributions of these enzymes may be explored by varying incubation conditions, such as pH. Presently, individual isoforms of carbonyl-reducing enzymes are not widely available, either in recombinant or purified form. However, it is possible to study carbonyl reduction clearance pathways from simple experiments with commercially available reagents. This article provides an overview of carbonyl-reducing enzymes, including some kinetic data for substrates and inhibitors. In addition, an experimental strategy for the study of these enzymes in vitro is presented.

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Carbonyl reducing enzymes are involved in the metabolism of endogenous as well as xenobiotic molecules. Enzymes that catalyze the reversible oxidoreduction of aldehyde and ketone moieties include alcohol dehydrogenases, aldo-keto reductases, quinone reductases, and short-chain dehydrogenases/reductases. These enzymes differ with respect to subcellular location, cofactor dependence, and susceptibility to chemical inhibitors. Thus, it is possible to assess the relative contributions of these enzyme systems in the hepatic metabolism of a particular xenobiotic through simple in vitro experiments with commercially available reagents. The approaches described in this unit assume the availability of analytical procedures for measuring the parent compound and metabolites, such as HPLC with radiochemical, UV, or MS detection. Thus, the purpose of this unit is to outline methods for the study of the enzymatic carbonyl reduction of a drug development candidate or other xenobiotic molecule of interest.

Maltose-binding protein (MBP), whose export in E. coli is dependent upon the chaperone SecB, and ribose-binding protein (RBP), whose export is SecB-independent, have been used to generate hybrid secretory proteins. Here, in vitro techniques were used to analyze MBP, RBP, RBP-MBP (RBP signal and MBP mature), and MBP-RBP (MBP signal and RBP mature). In protease-protection experiments, RBP folded considerably faster than MBP, RBP-MBP, or MBP-RBP. Only the folding properties of proteins containing the MBP mature moiety were influenced by SecB. In post-translational translocation assays, MBP exhibited the highest translocation efficiency. The hybrids RBP-MBP and MBP-RBP showed intermediate levels, and RBP translocation was not detected in these assays. These experiments demonstrate the influence of the signal peptide in determining folding properties and translocation efficiency of precursor secretory proteins.