A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997) Protein Eng. 10, 119 -129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b 5 . Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b 5 and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b 5 . All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b 5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.The cytochromes P450 (P450s) 1 are a ubiquitous superfamily of mixed function oxidases that catalyze the oxidation of a large number of hydrophobic endogenous and xenobiotic substrates. Known substrates number in the thousands, whereas unique P450 sequences are counted in the hundreds at this time (2-4).The versatility of these oxidases and their potential for industrial purposes has generated a great deal of interest in understanding their structure, function, and redox reactions. The reaction catalyzed by P450 is shown in Reaction 1.where RH is the substrate and ROH is the oxidized product.The enzymatic cycle includes substrate binding, first electron transfer, oxygen binding, second electron transfer, substrate oxidation, and finally product dissociation. The redox partners for the microsomal P450s are cytochrome P450 reductase (P450 reductase) which contains both a FAD and FMN cofactor and cytochrome b 5 (cyt b 5 ). The crystal structure of P450 reductase has recently been published, and the two domains of the enzyme have been individually expressed and characterized (5, 6). In contrast, the crystal structure of cyt b 5 has been known for many years but has just recently been refined (7,8). The first and second electrons are donated to P450 by P450 reductase. Because of its redox potential (Х ϩ 25 mV), cyt b 5 can only donate the second electron to P450 (9). In fact, it has been suggested that cyt b 5 may be able to transfer the second electron to selected P450s even faster than P450 reductase, thereby decreasing the amount of superoxide produced (1...

"New" glucose production has been measured in 54 infants and children for the first time by continuous three-to-four-hour influsion of the safe, nonradioactive tracer 6,6-dideuteroglucose. The use of combined gas chromatography--mass spectrometry with monitoring of selected ions allowed deuterium enrichment in blood glucose to be measured on microliter samples with an error of less than 2 per cent. In the young child, glucose production increased in a slightly curvilinear manner from 1 kg. to 25 kg. body weight, when it reached 140 mg. per minute, almost the adult value of 173 mg. per minute (2.28 +/- 0.23 mg./kg.-min., mean +/- S.E.). Normalized for weight, glucose production in premature infants was 5.46 +/- 0.31 mg./kg.-min., in term neonates averaged 6.07 +/- 0.27 mg./kg.-min., in children below the age of six years was 7.1 +/- 0.27 mg./kg.-min., and in late childhood averaged 5.4 +/- 0.28 mg./kg.-min. Relative to estimated brain weight, however, glucose production was essentially linear from the 1-kg. premature infant to the 80-kg. adult. These data, the first measurements of "new" glucose production in childhood, suggest that brain size may be a principal determinant of those factors that regulate hepatic glucose output throughout life.

Glucose is an important substrate for myocardial metabolism. This study was designed to determine the effect of circulating metabolic substrates on myocardial glucose extraction and to determine the metabolic fate of glucose in normal human myocardium. Coronary sinus and arterial catheters were placed in 23 healthy male volunteers. 16-"'CjGlucose was infused as a tracer in 10 subjects. 16-'CiGlucose and IU-_3Cilactate were simultaneously infused in the other 13 subjects. Simultaneous blood samples were obtained for chemical analyses of glucose, lactate, and free fatty acids and for the isotopic analyses of glucose and lactate. Glucose oxidation was assessed by measuring myocardial 4 CO2 production. The amount of glucose extracted and oxidized by the myocardium was inversely correlated with the arterial level of free fatty acids (r = -0.71; P < 0.0001). 20% (range, 0-63%) of the glucose extraction underwent immediate oxidation. Chemical lactate analysis showed a net extraction of 26.0±16.4%. However, isotopic analysis demonstrated that lactate was being released by the myocardium. In the 13 subjects receiving the dual-carbon-labeled isotopes, the lactate released was 0.09±0.04 ,mol/ml and 49.5±29.5% of this lactate was from exogenous glucose. This study demonstrates that the circulating level of free fatty acids plays a major role in determining the amount of glucose extracted and oxidized by the normal human myocardium. Only 20.1±19.4% of the glucose extracted underwent oxidation, and 13.0±9.0% of the glucose extracted was metabolized to lactate and released by the myocardium. Thus, 60-70% of the glucose extracted by the normal myocardium is probably stored as glycogen in the fasting, resting state.

The complete stoichiometry of the metabolism of the cytochrome b 5 (cyt b 5 )-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b 5 for its metabolism. Cyt b 5 invariably improved the efficiency of product formation. That is, in the presence of cyt b 5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide.With methoxyflurane, cyt b 5 addition always resulted in an increased rate of product formation, while with benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b 5 . Under some experimental conditions cyt b 5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b 5 depends on the net effect of the stimulatory and inhibitory effects of cyt b 5 . When total NADPH consumption is inhibited by cyt b 5 , the rapidly metabolized, highly coupled (Х50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (Х0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b 5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.