A rapid reaction study of anthranilate hydroxylase. Evidence for a catalytically important conformational change during slow initial turnover with anthranilate

Rapid reaction kinetics of the flavoprotein anthranilate hydroxylase from Trichosporon cutaneum were examined for reactions involving anthranilate, the native substrate. As was reported earlier for the nonhydroxylated substrate analogue, salicylate, some reactions in the first turnover with anthranilate occur slower than those in subsequent turnovers (Powlowski, J., Massey, V., and Ballou, D. P. (1989) J. Biol. Chem. 264, 5606-5612). Evidence is presented for slow conformational changes that occur both on binding of the aromatic ligand and on reduction of the enzyme. These changes are apparently important for rapid anthranilate binding to occur in turnovers subsequent to the first. Moreover, bound anthranilate is required for rapid reduction of enzyme-bound FAD by NADPH. Studies to probe the accessibility of reagents to modified flavins that had been incorporated into the apoenzyme indicate that anthranilate binding causes a conformational change in the protein, allowing increased access to the benzene ring moiety of the flavin. An unusual isotope effect with (R)-NADPD (4(R)-2H] NADPH) is observed on Kd rather than on kred, which is consistent with a model involving slow interconversion of enzyme-substrate complexes before productive binding of NADPH and reduction of the enzyme flavin.     

Effect of substrate and pH on the oxidative half-reaction of phenol hydroxylase

The oxidative half-reaction of phenol hydroxylase has been studied by stopped-flow spectrophotometry. Three flavin-oxygen intermediates can be detected when the substrate is thiophenol, or m-NH2, m-OH, m-CH3, m-Cl, or p-OH phenol. Intermediate I, the flavin C(4a)-hydroperoxide, has an absorbance maximum at 380-390 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate III, the flavin C(4a)-hydroxide, has an absorbance maximum at 365-375 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate II has absorbance maxima of 350-390 nm and extinction coefficients of 10,000-16,000 M-1 cm-1 depending on the substrate. A Hammett plot of the logarithm of the rates of the oxygen transfer step, the conversion of intermediate I to intermediate II, gives a straight line with a slope -0.5. Fluoride ion is a product of the enzymatic reaction when 2,3,5,6-tetrafluorophenol is the substrate. These results are consistent with an electrophilic substitution mechanism for oxygen transfer. The conversions of I to II and II to III are acid-catalyzed. A kinetic isotope effect of 8 was measured for the conversion of II to III using deuterated resorcinol as substrate. The conversion of III to oxidized enzyme is base-catalyzed, suggesting that the reaction depends on the removal of the flavin N(5) proton. Product release occurs at the same time as the formation of intermediate III, or rapidly thereafter. The results are interpreted according to the ring-opened model of Entsch et al. (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563).     

Decay of the 4a-hydroxy-FAD intermediate of phenol hydroxylase

The oxidative half-reaction of phenol hydroxylase involves the formation of three spectrally distinct intermediates (Detmer, K.M., and Massey, V. (1985) J. Biol. Chem. 260, 5998-6005). Addition of an aerobic NADPH-regenerating system, phenol, and azide quantitatively converted oxidized enzyme to the third intermediate, a 4a-hydroxy-FAD species (Detmer, K.M., and Massey, V. (1984) J. Biol. Chem. 259, 11265-11272). This intermediate was isolated in the presence of azide and a wide variety of phenolic ligands. Decay rates were followed for the dehydration of 4a-hydroxy-FAD enzyme resulting in the original oxidized form. Deviation from the rate observed in the absence of phenolic ligands was presumed to be indicative of a binding interaction. Several phenols displayed further stabilization of the 4a-hydroxyflavin species. These ligands exhibited saturation kinetics with respect to the decay half-lives, consistent with a mechanistic model in which both free and bound 4a-hydroxy-FAD enzyme may be directly dehydrated to produce the oxidized species. The lack of stabilization by catechol, the natural product, suggests that product is released from the enzyme during turnover by the time that this intermediate is formed. A pH profile, generated for the decay rates in the absence and presence of phenolic ligand, suggests both acid and base catalysis by hydronium ion and hydroxide, respectively.     

Absorption spectra of the hydroxycyclohexadienyl radicals of substrates for phenol hydroxylase

The absorption spectra of the hydroxycyclohexadienyl radicals formed upon the addition of OH radicals to six substrates for phenol hydroxylase have been determined using pulse radiolysis. Combining the radical spectra of thiophenol (lambda max, 390 nm; epsilon, 10,500 M-1 cm-1) and resorcinol (lambda max, 340 nm; epsilon, 4,100 M-1 cm-1) with their respective published spectra of enzyme-bound reduced flavin that is substituted in the C(4a) position of the dihydroflavin ring gave composite spectra that closely match the spectra formed concomitantly with the introduction of an oxygen atom into the substrates, the so-called Intermediate II species. A similar procedure for the substrates hydroquinone, 3-aminophenol, 3-chlorophenol, and 3-methylphenol yielded spectra that are also consistent with the known characteristics of their Intermediate II species. These spectral results give further support to the proposed biradical mechanism (Anderson, R.F., Patel, K. B., and Stratford, M. R. L. (1987) J. Biol. Chem. 262, 17475-17479) for the functioning of this class of flavoprotein hydroxylases.     

On the structure of flavin-oxygen intermediates involved in enzymatic reactions.

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421--505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C(4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the subsituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavoproteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides.     

Properties of anthranilate hydroxylase (deaminating), a flavoprotein from Trichosporon cutaneum

Anthranilate hydroxylase was purified from the yeast Trichosporon cutaneum. This enzyme is a simple flavoprotein which apparently does not require any additional cofactor for the conversion of anthranilate to 2,3-dihydroxybenzoate. Anthranilate hydroxylase has Mr of approximately 95,000, with subunit Mr of 50,000; it contains 2 mol of FAD/mol of enzyme. A number of compounds in addition to anthranilate serve as substrates, or effectors, for this enzyme. Oxygen-labeling experiments show that the oxygen atom at the 3-position of the product, 2,3-dihydroxybenzoate, originates from O2, while that at the 2-position is derived from H2O. A mechanism is proposed involving imine formation and hydrolysis during the reaction with the flavin hydroperoxide formed from reduced enzyme flavin and molecular oxygen. This proposal is in accord with the mechanism postulated for other flavoprotein aromatic hydroxylases.     

Molecular characterization of the 4'-phosphopantothenoylcysteine synthetase domain of bacterial dfp flavoproteins

In bacteria, coenzyme A is synthesized in five steps from pantothenate. The flavoprotein Dfp catalyzes the synthesis of the coenzyme A precursor 4'-phosphopantetheine in the presence of 4'-phosphopantothenate, cysteine, CTP, and Mg(2+) (Strauss, E., Kinsland, C., Ge, Y., McLafferty, F. W., and Begley, T. P. (2001) J. Biol. Chem. 276, 13513-13516). It has been shown that the NH(2)-terminal domain of Dfp has 4'-phosphopantothenoylcysteine decarboxylase activity (Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M., and Steinbacher, S. (2000) J. Biol. Chem. 275, 31838-31846). Here I demonstrate that the COOH-terminal CoaB domain of Dfp catalyzes the synthesis of 4'-phosphopantothenoylcysteine. The exchange of conserved amino acid residues within the CoaB domain revealed that the synthesis of 4'-phosphopantothenoylcysteine occurs in two half-reactions. Using the mutant protein His-CoaB N210D the putative acyl-cytidylate intermediate of 4'-phosphopantothenate was detectable. The same intermediate was detectable for the wild-type CoaB enzyme if cysteine was omitted in the reaction mixture. Exchange of the conserved Lys(289) residue, which is part of the strictly conserved (289)KXKK(292) motif of the CoaB domain, resulted in complete loss of activity with neither the acyl-cytidylate intermediate nor 4'-phosphopantothenoylcysteine being detectable. Gel filtration experiments indicated that CoaB forms dimers. Residues that are important for dimerization are conserved in CoaB proteins from eubacteria, Archaea, and eukaryotes.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris.

Saccharomyces SUC2 invertase, secreted by the methylotrophic yeast Pichia pastoris and purified to homogeneity from the growth medium by DE-52 chromatography, appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a diffuse ladder of species at 85-90 kDa, while the secreted Saccharomyces form migrated as a broad band from 100 to 150 kDa. Endo-beta-N-acetylglucosaminidase H released the Pichia invertase carbohydrate generating a 60-kDa protein with residual Asn-linked GlcNAcs and oligosaccharides separated on Bio-Gel P-4 into Man8-11GlcNAc. Nearly 75% of the oligosaccharides were equally distributed between Man8,9GlcNAc, while 17% were Man10GlcNAc and 8% were Man11GlcNAc. Oligosaccharide pools were analyzed for homogeneity by high-pH anion-exchange chromatography, and structures were assigned using 500 MHz one- and two-dimensional 1H NMR spectroscopy. Pichia Man8GlcNAc was the same isomer as found in Saccharomyces, which arises by removing the alpha 1,2-linked terminal mannose from the middle arm of the lipid-oligosaccharide Man9GlcNAc (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). The Man9GlcNAc pool was 5% lipid-oligosaccharide precursor and 95% Man8GlcNAc isomer with a terminal alpha 1,6-linked mannose on the lower-arm alpha 1,3-core-linked residue (Hernández, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). An alpha 1,2-linked mannose on the new alpha 1,6-linked branch in Man9GlcNAc provided 80% of the Man10GlcNAc, which is the structure on Saccharomyces invertase (Trimble, R. B., and Atkinson, P. H. (1986) J. Biol. Chem. 261, 9815-9824). A minor Man10GlcNAc (12%) and the principal Man11GlcNAc (82%) were the major Man9,10GlcNAc with novel alpha 1,2-linked mannoses on the preexisting alpha 1,2-linked termini. Although Pichia glycans did not have terminal alpha 1,3-linked mannoses as found on Saccharomyces core oligosaccharides, over 60% of the structures were isometric configurations unique to lower eukaryotes.     

Synergistic interactions of multiple mutations on catalysis during the hydroxylation reaction of p-hydroxybenzoate hydroxylase: studies of the Lys297Met, Asn300Asp, and Tyr385Phe mutants reconstituted with 8-Cl-flavin

The oxygen transfer to p-hydroxybenzoate catalyzed by p-hydroxybenzoate hydroxylase (PHBH) has been shown to occur via a C4a-hydroperoxide of the flavin. Two factors are likely to be important in facilitating the transfer of oxygen from the C4a-hydroperoxide to the substrate. (a) The positive electrostatic potential of the active site partially stabilizes the negative charge centered on the oxygen of the flavin-C4a-alkoxide leaving group during the transition state [Ortiz-Maldonado, M., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 8124-8137]. (b) The hydrogen-bonding network ionizes the substrate to promote its nucleophilic attack on the electrophilic C4a-hydroperoxide intermediate [Entsch, B., Palfey, B. A., Ballou, D. P., and Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. This ionization is also aided by the positive electrostatic potential of the active site [Moran, G. R., Entsch, B., Palfey, B. A., and Ballou, D. P. (1997) Biochemistry 36, 7548-7556]. Substituents on the flavin can specifically affect the stability of the alkoxide leaving-group, whereas changes to specific enzyme residues can affect the charge in the active site and the hydrogen-bonding network. We have used wild-type (WT) PHBH and several mutant forms, all with normal FAD and with 8-Cl-FAD substituted for FAD, to assess the relative contributions of the two effects. Lys297Met and Asn300Asp have decreased positive charge in the active site, and these variants engender approximately 35-fold slower hydroxylation rates than the WT enzyme. Substitution of 8-Cl-FAD in these mutant forms gives approximately 1.8-fold increases in hydroxylation rates, compared with a > or =4.8-fold increase for WT with this flavin. The hydroxylation catalyzed by Tyr385Phe, a mutant enzyme form with a disrupted hydrogen-bonding network that compromises the ionization of the substrate without changing the positive charge of the active site, is stimulated 1.5-fold by substituting the enzyme with 8-Cl-FAD. The substrate, tetrafluoro-p-hydroxybenzoate, is fully ionized in WT PHBH, but this phenolate is a poor nucleophile because of the electron-withdrawing effects of the fluorine substituents. With tetrafluoro-p-hydroxybenzoate as the substrate, substitution of FAD with 8-Cl-FAD in the WT enzyme stabilizes the leaving alkoxide and leads to a 2.3-fold increase in the hydroxylation rate compared to that with FAD. Either the use of substrates that do not communicate with the proton network or the mutation of amino acid residues that perturb this interaction may prevent a necessary conformational change that allows proper orientation between reactants during the hydroxylation reaction or permits the essential protonation of the initially formed nascent flavin-C4a-peroxide anion. Thus, both activation of substrate by the proton network and stabilization of the leaving alkoxide appear to be important for oxygen transfer catalyzed by PHBH. The full effect of the substituents on the flavin (4.8-fold) can only be realized when the optimal transition state can be achieved, and this optimal state is not fully realized with the mutant forms.     

Subunit exchange between membranous and soluble forms of bovine dopamine beta-hydroxylase

Dopamine beta-hydroxylase is present in the bovine adrenal medulla in two forms: soluble and membrane-bound. In a previous study, it was shown that the tetrameric, soluble form of the enzyme undergoes dissociation into two identical dimeric subunits and that this subunit dissociation is dependent on pH and ADP binding (Dhawan, S., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1986) J. Biol. Chem. 261, 7680-7684). Here we report the effect of pH and ADP on the dissociation of the membranous form of dopamine beta-hydroxylase into two nonidentical subunits. Negative stain electron microscopy of purified membranous hydroxylase showed largely tetrameric species together with occasional dimeric species. The tetrameric images of membranous hydroxylase were similar to, but clearly different from, previously published negative stain images of soluble hydroxylase (Duong, L. T., Fleming, P. J., and Ornberg, R. L. (1985) J. Biol. Chem. 260, 2393-2398). Quantitative binding of ADP to the membranous hydroxylase revealed the existence of two binding sites per dimeric subunit. ADP binding and low pH both promote dissociation of a hydrophilic, catalytically active subunit from the membranous enzyme reconstituted onto phospholipid vesicles. Kinetic analyses of reconstituted membranous hydroxylase activity were consistent with the existence of tetrameric and dimeric catalytic species in equilibrium. All of the hydrophilic subunits of the purified soluble hydroxylase bind to the hydrophobic subunits of the reconstituted membranous hydroxylase. We propose that, in the chromaffin granules, the soluble hydroxylase subunits are in equilibrium association with the membrane-bound hydroxylase subunits and that the hydrophilic subunits of both soluble and membranous hydroxylase are identical.     

The mnn2 mutant of Saccharomyces cerevisiae is affected in phosphorylation of N-linked oligosaccharides

We studied the phosphorylation of the inner core region of N-linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain. We performed structural studies of the N-oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 [Hernández, L.M., Ballou, L., Alvarado, E., Tsai, P.-K. and Ballou, C.E. (1989) J. Biol. Chem. 264, 13648-13659]. We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A(3) (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C(1)) is allowed, although it is also reduced. *Phosphorylation sites in mnn1mnn9. (see structure below)     

pH-jump studies at subzero temperatures on an intermediate in the reaction of xanthine oxidase with xanthine.

Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at--82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to--60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.     

A novel two-protein component flavoprotein hydroxylase.

p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) was purified from Acinetobacter baumannii and shown to be a two-protein component enzyme. The small component (C1) is the reductase enzyme with a subunit molecular mass of 32 kDa. C1 alone catalyses HPA-stimulated NADH oxidation without hydroxylation of HPA. C1 is a flavoprotein with FMN as a native cofactor but can also bind to FAD. The large component (C2) is the hydroxylase component that hydroxylates HPA in the presence of C1. C2 is a tetrameric enzyme with a subunit molecular mass of 50 kDa and apparently contains no redox centre. FMN, FAD, or riboflavin could be used as coenzymes for hydroxylase activity with FMN showing the highest activity. Our data demonstrated that C2 alone was capable of utilizing reduced FMN to form the product 3,4-dihydroxyphenylacetate. Mixing reduced flavin with C2 also resulted in the formation of a flavin intermediate that resembled a C(4a)-substituted flavin species indicating that the reaction mechanism of the enzyme proceeded via C(4a)-substituted flavin intermediates. Based on the available evidence, we conclude that the reaction mechanism of HPAH from A. baumannii is similar to that of bacterial luciferase. The enzyme uses a luciferase-like mechanism and reduced flavin (FMNH2, FADH2, or reduced riboflavin) to catalyse the hydroxylation of aromatic compounds, which are usually catalysed by FAD-associated aromatic hydroxylases.     

Point mutations at the type I Cu ligands, Cys457 and Met467, and at the putative proton donor, Asp105, in Myrothecium verrucaria bilirubin oxidase and reactions with dioxygen

The type I Cu site in the Cys457Ser mutant of Myrothecium verrucaria bilirubin oxidase was vacant, but the trinuclear center composed of a type II Cu and a pair of type III Cu's was fully occupied by three Cu ions. Cys457Ser could react with dioxygen, affording reaction intermediate I with absorption maxima at 340, 470, and 675 nm. This intermediate corresponds to that obtained from laccase, whose type I Cu is cupric and type II and III Cu's are cuprous [Zoppellaro, G., Sakurai, T., and Huang, H. (2001) J. Biochem. 129, 949-953] or whose type I Cu is substituted with Hg [Palmer, A. E., Lee, S. K., and Solomon, E. I. (2001) J. Am. Chem. Soc. 123, 6591-6599]. Another type I Cu mutant, Met467Gln, with modified spectroscopic properties and redox potential, afforded reaction intermediate II with absorption maxima at 355 and 450 nm. This intermediate corresponds to that obtained during the reaction of laccase [Sundaram, U. M., Zhang, H. H., Hedman, B., Hodgson, K. O., and Solomon, E. I. (1997) J. Am. Chem. Soc. 119, 12525-12540; Huang, H., Zoppellaro, G., and Sakurai, T. (1999) J. Biol. Chem. 274, 32718-32724]. According to a three-dimensional model of bilirubin oxidase, Asp105 is positioned near the trinuclear center. Asp105Glu and Asp105Ala exhibited 46 and 7.5% bilirubin oxidase activity compared to the wild-type enzyme, respectively, indicating that Asp105 conserved in all multi-copper oxidases donates a proton to reaction intermediates I and II. In addition, this amino acid might be involved in the formation of the trinuclear center and in the binding of dioxygen based on the difficulties in incorporating four Cu ions in Asp105Ala and Asp105Asn and their reactions with dioxygen.     

Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (lambda max = 500 nm) relative to that of native enzyme (lambda max = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, deadend complex formed by each analog is significantly blue-shifted (lambda max less than 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.     

Sequence of the small subunit of yeast carbamyl phosphate synthetase and identification of its catalytic domain

The yeast gene CPA1 coding for the small subunit of arginine-specific carbamyl phosphate synthetase has been cloned by complementation of a cpa 1 mutant with a plasmid library of total yeast chromosomal DNA. Two of the plasmids, pJL113/ST4 and pJL113/ST15, contain DNA inserts in opposite orientations with overlapping sequences of 2.6 kilobases. The nucleotide sequence of a 2.2-kilobase region of the DNA insert carrying the CPA1 gene has been determined. The CPA1 gene has been identified to be 1233 nucleotides long and to code for a polypeptide of 411 amino acids with a calculated molecular weight of 45,358. The amino acid sequence encoded in CPA1 is homologous to the recently determined sequence of the small subunit of Escherichia coli carbamyl phosphate synthetase (Piette, J., Nyunoya, H., Lusty, C.J., Cunin, R., Weyens, G., Crabeel, M., Charlier, D., Glandsdorff, N., and Pierard, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4134-4138) over the entire length of the polypeptide chain. Comparison of the amino acid sequences of the small subunits of yeast and E. coli carbamyl phosphate synthetases to the sequences of Component II of anthranilate and p-aminobenzoate synthases suggests that these amidotransferases are evolutionarily related. The most highly conserved region of the yeast and E. coli enzymes includes a cysteine residue previously found to be at the active site of Pseudomonas putida anthranilate synthase Component II (Kawamura, M., Keim, P.S., Goto, Y., Zalkin, H., and Heinrikson, R.L. (1978) J. Biol. Chem. 253, 4659-4668). Based on the observed homologies in the primary sequences of the other amidotransferases examined, we propose a 13-amino acid long sequence to be part of the catalytic domain of this class of enzymes.     

Absolute stereochemistry of flavins in enzyme-catalyzed reactions

The 8-demethyl-8-hydroxy-5-deaza-5-carba analogues of FMN and FAD have been synthesized. Several apoproteins of flavoenzymes were successfully reconstituted with these analogues. This and further tests established that these analogues could serve as general probes for flavin stereospecificity in enzyme-catalyzed reactions. The method used by us involved stereoselective introduction of label on one enzyme combined with transfer to and analysis on a second enzyme. Using as a reference glutathione reductase from human erythrocytes for which the absolute stereochemistry of catalysis is known from X-ray studies [Pai, E. F., & Schulz, G. E. (1983) J. Biol. Chem. 258, 1752-1758], we were able to determine the absolute stereospecificities of other flavoenzymes. We found that glutathione reductase (NADPH), general acyl-CoA dehydrogenase (acyl-CoA), mercuric reductase (NADPH), thioredoxin reductase (NADPH), p-hydroxybenzoate hydroxylase (NADPH), melilotate hydroxylase (NADH), anthranilate hydroxylase (NADPH), and glucose oxidase (glucose) all use the re face of the flavin ring when interacting with the substrates given in parentheses.     

Biosynthesis of cholestanol from bile acid intermediates in the rabbit and the rat

Biliary 7 alpha-hydroxy-4-cholesten-3-one (an intermediate in bile acid biosynthesis) may be 7 alpha-dehydroxylated in the gut and further metabolized to cholestanol (Skrede, S., and Bj?rkhem, I. (1982) J. Biol. Chem. 257, 8363-8367). We have now evaluated the quantitative importance of pathway(s) to cholestanol with 7 alpha-hydroxylated C27 steroids as intermediates. After feeding conventionally fed rabbits or rats or germ-free rats with [7 alpha-3H]cholesterol and [4-14C]cholesterol, tissue cholestanol could be isolated with about a 20% lower 3H/14C ratio than present in cholesterol. We conclude that there is a pathway to cholestanol involving 7 alpha-hydroxylated intermediates. Intestinal microorganisms are not essential for this pathway, which accounts for at most 20% of the cholestanol formed in these species. In bile fistula rats, there was also a significant conversion of intraperitoneally injected [7 beta-3H]7 alpha-hydroxycholesterol and [4-14C]7 alpha-hydroxy-4-cholesten-3-one into cholestanol. The enzymes involved in the 7 alpha-hydroxylation/dehydroxylation pathway for the biosynthesis of cholestanol are probably located in the liver. Both 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one may be intermediates.     

Spectroscopic evidence for an intermediate in the T6 to R6 allosteric transition of the Co(II)-substituted insulin hexamer.

The phenol-induced conformational transition in the insulin hexamer is known to involve a large change in structure wherein residues 1-8 of the insulin B-chain are transformed from an extended coil (T-state) to a helix (R-state). This change in protein conformation both exposes a cryptic protein pocket on each subunit to which phenol binds and forces the HisB10 zinc sites to undergo a change in coordination geometry from octahedral to tetrahedral [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 593-596]. Substitution of Co(II) for Zn(II) at the HisB10 sites introduces a sensitive chromophoric probe of the structural and chemical events that occur during this allosteric transition [Roy, M., Brader, M. L., Lee, R. W.-K., Kaarsholm, N. C., Hansen, J. F., & Dunn, M. F. (1989) J. Biol. Chem. 264, 19081-19085]. In this study, using rapid-scannig stopped-flow (RSSF) UV-visible spectroscopic studies, we demonstrate that a transient chemical intermediate is formed during the phenol-induced conversion of Co(II)-substituted hexamer from the T-state to the R-state. Decomposition of the RSSF spectra gave a spectrum for the intermediate with d-d transitions consistent with the assignment of the intermediate as either a distorted tetrahedral or a 5-coordinate Co(II) species. Possible structures for the intermediate and the implications of these findings to the allosteric mechanism are considered.