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The enzyme has been prepared from Aerobacter aerogenes and from Escherichia coli,
and is composed of two apparently identical subunits. Kinetic studies have been made
of the mechanisms for the two separate reactions and of the overall reaction. These and
other data are being used to determine the relationship between the sites for catalysis of
the two reactions.
The steady-state kinetics indicate that the two reactions could occur either at one
active site or at two separate sites with similar kinetic properties (Heyde & Morrison,
1978). Thus two values for the dissociation constant for the interaction of prephenate
with the free enzyme, determined by its function as an inhibitor of the mutase reaction
or a substrate of the dehydrogenase reaction, are similar. So also are the pairs of dissociation constants for the interaction of NAD+ or NADH with the free enzyme, determined by their function in the dehydrogenase reaction or as activators of the mutase
reaction. However, the values determined for the dissociation constant of the product
hydroxyphenylpyruvate, which inhibits both reactions, appear to differ significantly.
Among the approaches being used to determine the relationship between the sites
for the two reactions is chemical inactivation of the enzyme. A variety of inactivating
conditions lead to parallel loss of the two enzymic activities, and the inactivation by
iodoacetamide was studied in detail. All reactants afford some protection against this
reagent, and from the protective effects a dissociation constant was determined for each
reactant. The values are consistent with the kinetically determined dissociation constants for prephenate and the coenzyme, whereas for hydroxyphenylpyruvate the value
obtained is similar to the kinetic constant from the mutase reaction rather than that
from the dehydrogenase reaction. It appears possible that the kinetic analysis may be
complicated by the resemblance between hydroxyphenylpyruvate and tyrosine, the
allosteric end-product inhibitor. Tyrosine itself causes rather weak protection of both
activities, as does NAD+, but when both these compounds are present complete protection is observed over a time period during which 99% of unprotected activity is lost.
This effect is consistent with the marked enhancement by NAD+ of the directly measured
binding of tyrosine.
The protection results cannot provide definitive evidence that the two reactions occur
at one active site. However, they indicate a close relationship between the sites for the
two activities. This is consistent with previous observations that some hydroxyphenylpyruvate can be produced from chorismate without equilibration of the intermediate
prephenate with the medium (Heyde, 1979).
Heyde, E. (1979) Biochemistry in the press
Heyde, E. & Morrison, J. F. (1978) Biochemistry 17, 1573-1580
Microsomal Biphenyl Hydroxylation:The Effect of Selective Deuterium
Substitution on the Rate of Formation of the Monohydroxybiphenyls
JAMES W. BRIDGES and DAVID McKILLOP
Department of Biochemistry, University of Surrey, Guildford, Surrey G U 2 5 XH, U.K.
and PETER J. COX, PETER B. FARMER, ALLAN B. FOSTER and
MICHAEL JARMAN
Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer
Hospital, Fulham Road, London SW3 6JB, U.K.
When C-H bond cleavage is the rate-determining step in a chemical transformation,
replacement of hydrogen by deuterium will result in an isotope effect. When isotope
effects occur in metabolic processes, the appropriate replacement of hydrogen by
deuterium will cause retardation, and, if alternative pathways exist, metabolism may
be diverted. This phenomenon has been termed ‘metabolic switching’ (Horning et a].,
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BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. Effect of selective deuteration on the rate of monohydroxylation of biphenyl by
rat liver microsomal fraction
Biphenyl and 3,3‘,5,5’-’H4- and 2,2’,4,4’,6,6’-2H6-1abelled
variants were incubated with
rat liver microsomal fraction from non-induced (control) or induced rats, and yields
of 2-, 3- and 4-hydroxybiphenyl were determined as described in the text. The results
are expressed as mean f S.E.M. ( n = 3). P values were determined by Student’s t test,
and an asterisk (*) denotes differences with P<O.O5.
Rats from which
microsomal
Rate of hydroxylation (pmol/min per mg of protein)
fraction was
,
prepared
Biphenyl 2-Hydroxylation 3-Hydroxylation 4-Hydroxylation
Control
Unlabelled
3725
20f4
443 f 42
’H,-labelled
68 k 8*
34f 1*
352 f 39
’H6-labeIled
36+2
16f3
488 f 28
PhenobarbiUnlabelled
47+4
793f115
114k 15
tone-induced ’H4-labelled
7 6 5 1*
124f6
680 f 44
’H6-label1ed
49+2
105f 5
888 f 66
3-MethylchoUnlabelled
272 f 22
130f 10
711 k 6 4
lanthreneZH4-labelled
232 f 20
506 54
110f 12
induced
’H~-hbekd
277 k 16
110f 10
654 f 91
+
1975), and has been exemplified in studies of selectively deuterated analogues of antipyrine and caffeine (Horning et al., 1975)) and of the antitumour agent 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea(‘CCNU’) (Farmer et al., 1978). Perdeuteration of
biphenyl produces a significant isotope effect (kH/ko= 1.27-1.45) on 3-hydroxylation
by liver microsomal preparations from the mouse, the rat and the hamster (Billings &
McMahon, 1978). It was suggested that 3-hydroxylation occurs at least in part by
direct oxygen insertion, a mechanism expected to produce an isotope effect, but that
2- and 4-hydroxylation, for which no isotope effect was observed, proceed via the
conventional aromatic hydroxylation mechanism of arene oxide formation followed by
rearrangement. On the basis of these studies, and of those concerned with metabolic
switching, we predicted that [3,3’,5,5’-2H4]biphenyl would be less rapidly 3-hydroxylated than biphenyl, and that hydroxylation would in consequence be switched towards
the 2- and the 4-positions. In contrast, [2,2’,4,4’,6,6’-’H6]biphenyl,
in which there is no
isotopic substitution known to elicit an isotope effect, should yield the same metabolism
profile as biphenyl. To test this hypothesis, the rates of formation of the various monohydroxy derivatives from biphenyl and the two deuterated analogues (Akawie et al.,
1959) in the presence of rat liver microsomal fraction were compared.
Male Wistar albino rats (1OOg) were injected intraperitoneally for two consecutive
days withphenobarbitone(80mg/kg in saline) or 3-methylcholanthrene(25 mg/kg in corn
oil), and the hepatic microsomal fraction was prepared by differential ultracentrifugation
as described previously (McPherson et a/., 1976). Microsomal fraction was also prepared from non-induced animals. The biphenyl and deuterated analogues, added in
dimethylformamide (lopl), were incubated (concn. 1 mM) with microsomal fraction
and cofactors, and the products were recovered and analysed by high-pressure liquid
chromatography as described previously (Burke & Prough, 1977; Burke et al., 1977).
The results (Table 1) accord only partly with prediction. Thus there was, as expected,
no difference in the rates of hydroxylation at any of the three sites between biphenyl
and the ‘H,-labelled analogue. Also, the predicted increase in the rate of 2-hydroxylation for the 2H,-labelled analogue was observed in the experiments with microsomal
fraction from non-induced or phenobarbital-induced animals. However, this was not a
1979
583rd MEETING. CAMBRIDGE
1075
simple consequence of metabolic switching, since the predicted retardation of 3-hydroxylation for the ZH4-labelledanalogue did not occur. Indeed, 3-hydroxylation was actually
more rapid for this analogue than for biphenyl when microsomal-fraction from noninduced animals was used. Moreover, the predicted increase in 4-hydroxylation for the
ZH4-labelledanalogue did not occur; indeed, there was a trend towards a lower rate for
4-hydroxylation of this analogue compared with the ZH,-labelled and the non-deuterated forms.
A possible explanation for the above anomalies may be that deuterium substitiuton
might slightly alter, and to various extents, the affinity of the substrate for the different
microsomal mono-oxygenases. The steric requirement of hydrogen appears to be
significantly more than that of deuterium (Lee et al., 1978). Deuterium substitution
can also change lipophilicity, as evidenced by the ability of high-pressure liquid chromatography to resolve deuterated compounds from their unlabelled counterparts (see, e.g.,
Farmer et al., 1978). Such an explanation could account for species differences in the
isotope effect for 3-hydroxylation of biphenyl (Billings & McMahon, 1978) and the
dependence on the method of induction (Table 1) of the relative rates of hydroxylation
at the various sites of the biphenyl variants used in the present study. To test this second
hypothesis, further studies on the effect of selective deuteration on hydroxylation of
biphenyl need to be carried out with the separate components of the microsomal monooxygenase system.
Akawie, R. I., Scarborough, J. M. & Burr, J. G. (1959) J. Org. Chem. 24,946-949
Billings, R. E. & McMahon, R. E. (1978) Mol. Pharmucol. 14,145-154
Burke, M . D. & Prough, R. A. (1977) Anal. Biochem. 83,46&473
Burke, M. D., Benford, D. J., Bridges, J. W. & Parke, D. V. (1977) Biochem. SOC.
Trans. 5,
1370-1372
Farmer, P. B., Foster, A. B., Jarman, M., Oddy, M. R. & Reed, D. J. (1978)J. Med. Chem. 21,
514-520
Homing, M. G . , Haogele, K . D., Sommer, K. R., Nowlin, J., Stafford, M. & Thenot, J. P.
(1975) Proc. Int. Symp. Stable Isoropes 2nd. (Klein, E. R. & Klein, P. D., eds.), pp. 41-54,
National Technical Information Service, U S . Department of Commerce, Springfield
Lee,S.-Y.,Rarth, G., Kieslich, K. & Djerassi, C. (1978)J. Am. Chem. SOC.
100,3965-3966
McPherson, F. J., Bridges, J. W. & Parke, D. V. (1976) Biochem. Pharmacol.25,1345-1350
Evidence for a Model of Regeneration of a Protonated Species, bR, from
a Phototransient, M, in the Photochemical Cycle of Bacteriorhodopsin
from Halobacterium halobium
MARY E. EDGERTON and COLIN GREENWOOD
School of Biological Sciences, Unfversity of East Anglia, Norwich NR4 7TJ, U.K.
The purple membrane of Halobacterium halobium contains a single protein bound via
a Schiff base to a retinal (Oesterhelt et a!., 1973; Lewis et al., 1974). This proteinretinal complex, bacteriorhodopsin, undergoes a light-driven reaction cycle that
results in a vectorial flow of protons across the membrane (Oesterhelt & Stoeckenius,
1973). The photocycle can be described by the following sheme, defined by Lozier et al.
(1979, the superscripts denoting the approximate wavelength maxima (nm) :
Studies carried out on species M show that it has a deprotonated Schiff base (Lewis et al.,
1974) and that, simultaneous with its decay, a proton is released from the membrane to
the exterior medium (Lozier et al., 1975). Decay of species M is not always monophasic,
as was once thought, but shows biphasicity under conditions of low temperature and
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