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S Ghisla

Publications and source records attributed to S Ghisla.

At least 127 records · Page 7Linked to original sources
PubMed1979

Structure of the flavin adduct formed in the suicide reaction of alpha-hydroxybutynoate with D-lactate dehydrogenase.

The Zn-dependent flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii is irreversibly inactivated by the D form of the suicide substrate 2-hydroxy-3-butynoic acid. The process of inactivation involves formation of a new pink chromophore, which can be released in intact form from the protein and which was purified to homogeneity by affinity chromatography. Inactivation involves covalent addition of the suicide substrate to the flavin coenzyme. The optical spectra indicate an elongation of the flavin chromophore, and the chemical reactivity suggests a derivative of reduced flavin. The structure of this adduct was deduced from Fourier transform NMR, from the chemical properties, and from comparison with appropriate models, which were synthesized chemically. This structure involves the covalent linkage of the acetylenic inhibitor to positions N(5) and C(6) of the flavin coenzyme via carbon atoms 2 and 4 of the inhibitor to form an additional fused aromatic ring. The pink adduct can be reconverted to an isoalloxazine chromophore by reduction with borohydride and subsequent reoxidation with oxygen. This new isoalloxazine has the spectral properties of an isoflavin, and it is proposed to carry the moiety of the inactivator molecule as substituent at position C(6). The structure of the pink chromophore representing a cyclic adduct to the flavin positions N(5) and C(6) is compared to that of the adduct obtained from L-lactate oxidase from Mycobacterium smegmatis and the L form of the same inhibitor [C(4a)--N(5) cyclic adduct; Schonbrunn, A., Abeles, R. H., Walsh, C. T., Ghisla, S., Ogata, H., and Massey, V. (1976) Biochemistry 15, 1978]. This comparison allows deductions about the relative orientation of substrate, coenzyme, and active center functional groups in the two enzymes.

Alkynes↗
PubMed1979

8-Mercaptoflavins as active site probes of flavoenzymes.

Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.

Binding Sites↗
PubMed1979

Properties of flavins where the 8-methyl group is replaced by mercapto- residues.

Sulfur functions in position 8 of the flavin nucleus give rise to new modified flavin derivatives, which should prove useful as probes of the flavin binding domains of flavoproteins. Here, we report on some properties of 8-nor-8-alkylmercaptoflavins and 8-nor-8-mercaptoflavin which are readily formed by nucleophilic displacement by alkylmercaptides or sulfide, with 8-nor-8-chloroflavins as starting material. The new flavins are characterized by extensive shifts in spectral properties, with very high extinction coefficients. 8-nor-8-mercaptoriboflavin is easily and reversibly converted to its (-S-S-) dimer. Oxidation of the sulfur group by peracids forms first sulfoxides and then sulfones, in which the characteristic usual flavin spectrum is regained. A comparison of 8-SR-8-nor-flavins with 8-OR-8-nor-flavins (Ghisla, S., and Mayhew, S.G. (1976) Eur. J. Biochem 63, 373-390) indicates that in both classes of compounds, optical properties, ionization constants, and oxidation-reduction potentials follow similar patterns.

Disulfides↗
PubMed1978

Biosynthesis of riboflavin. 6,7-Dimethyl-8-ribityllumazine 5'-phosphate is not a substrate for riboflavin synthase.

Phosphotransferase from carrot is shown to catalyze the phosphorylation of 6,7-dimethyl-8-ribityllumazine specifically at position 5' of the ribityl side chain. The lumazine 5'-phosphate is neither a substrate nor an inhibitor of riboflavin synthase from Bacillus subtilis and Escherichia coli. It follows that the obligatory product of riboflavin synthase is riboflavin and not FMN.

Bacillus subtilis↗
PubMed1977

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.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1976

Kinetic studies on the inactivation of L-lactate oxidase by [the acetylenic suicide substrate] 2-hydroxy-3-butynoate.

2-Hydroxy-3-butynoate is both a substrate and an irreversible inactivator of the flavoenzyme L-lactate oxidase. The partitioning between catalytic oxidation of 2-hydroxy-3-butynoate and inactivation of the enzyme is determined by the concentration of the second substrate, O2. Rapid reaction studies show the formation of an intermediate which is common to both the oxidation and inactivation pathways. This intermediate appears to be a charge-transfer complex between enzyme-reduced flavin and 2-keto-3-butynoate. It is characterized by a long-wavelength absorbing band (gamma(max) 600 nm) and lack of fluorescence, making it easily distinguished from the subsequently formed inactivated enzyme, which has no long wavelength absorption (gamma(max) 318, 368 nm) and which is strongly fluorescent. Inactivation is also accomplished by reaction of the reduced enzyme with 2-keto-3-butynoate. The absorbance and fluorescence characteristics of the inactivated enzyme are similar to those of a model compound, C(4a), N(5)-propano-bridged FMN bound to apolactate oxidase. That the modified chromophore of the inactivated enzyme is an adduct involving both the C(4a) and N5 positions is further supported by the spectral and fluorescence changes resulting from treatment of the inactivated enzyme with borohydride.

Anaerobiosis↗
PubMed1976

The structure of the covalent flavin adduct formed between lactate oxidase and the suicide substrate 2-hydroxy-3-butynoate.

2-Hydroxy-3-butynoic acid is a suicide substrate for Mycobacterium smegmatis lactate oxidase. Inactivation occurs by covalent modification of enzyme-bound FMN and does not involve labeling of the apoprotein. The spectrum of the enzyme bound adduct suggests that it is a 4a, 5-dihydroflavin derivative. When this adduct is released from the enzyme, a complex mixture of unstable compounds is obtained. When the initially formed enzyme-bound adduct is reduced with NaBH4, a major stable species can be resolved from the enzyme and can be isolated and purified. The structure was established by appropriate isotope substitutions. Fourier transform NMR spectroscopy, chemical reactivity, and synthesis of a model compound. The structure of the isolated adduct is structure II, Scheme II. The structure proposed for the adduct initially formed on the enzyme is structure VII, Scheme II.

Binding Sites↗
PubMed1976

Identification and properties of 8-hydroxyflavin--adenine dinucleotide in electron-transferring flavoprotein from Peptostreptococcus elsdenii.

1. A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenni. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine: (see article). Proof of this structure has been obtained by chemical syntehsis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methy-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (xi = 41 000 M-1 CM-1) and a pK at 4.8 due to the ionisation of the C(8)-OH group. 2. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN. 3. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form.

Binding Sites↗
PubMed1975

Mechanism of inactivation of the flavoenzyme lactate oxidase by oxalate.

Lactate oxidase from Mycobacterium smegmatic is strongly inhibited by oxalate. The inhibition is reversible in the absence and irreversible in the presence of light. Oxalate is bound to the enzyme in a two-step process with an over-all Kd of 1.6 times 10--5 M. The first step is a fast second order reaction with k-1/k1 equals 8.3 times 10--3 M, leading to a Complex I. This complex is then reversibly converted to a different Complex II in a slow first order reaction (k2 equals 40 min--1; k-2 equals 0.07 min--1), which is accompanied by major spectral perturbations of the flavin spectrum. With oxamate, two steps could not be demonstrated, and its binding is described by a single step reversible process, which is second order in oxamate (k1 equals 6.8 times 10-3 M--1 s--1, k-1 equals 28 s--1). Upon illumination the enzyme-oxalate Complex II is converted very rapidly at position N(5) of the flavin. Slow hydrolysis in the dark under anaerobic conditions subsequently yields free reduced enzyme. The light reaction of the oxamate comples is, in contrast, very slow and yields a stable N(5) urea adduct of the reduced flavin.

Binding Sites↗