Thermodynamics of loop formation in the denatured state of rhodopseudomonas palustris cytochrome c': scaling exponents and the reconciliation problem.

The observation that denatured proteins yield scaling exponents, nu, consistent with random-coil behavior and yet can also have pockets of residual or nonrandom structure has been termed the "reconciliation problem". To provide greater insight into the denatured state of a foldable sequence, we have measured histidine-heme loop formation equilibria in the denatured state of a class II c-type cytochrome, cytochrome c' from Rhodopseudomonas palustris. We have prepared a series of variants that provide His-heme loop stabilities, pK(loop)(His), for loop sizes ranging from 10 to 111 residues at intervals of 7 to 11 residues along the sequence of the protein. We observe a scaling exponent for loop formation, nu(3), of 2.5+/-0.3. Theoretical values for nu(3) range from 1.8 to 2.4; thus, the observed nu(3) is consistent with random-coil behavior. However, in contrast to data for loop formation as a function of loop size obtained with peptides of homogeneous sequence, we observe considerable scatter about the linear dependence of loop stability on loop size. Thus, foldable sequences behave very differently from homogeneous peptide sequences. The observed scatter suggests that there is considerable variation in the conformational properties along the backbone of a foldable sequence, consistent with alternating compact and extended regions. With regard to the reconciliation problem, it is evident that a scaling exponent consistent with a random coil is necessary but not sufficient to demonstrate random-coil behavior.

The effect of a Glu370Asp mutation in glutaryl-CoA dehydrogenase on proton transfer to the dienolate intermediate.

We have determined steady-state rate constants and net rate constants for the chemical steps in the catalytic pathway catalyzed by the E370D mutant of glutaryl-CoA dehydrogenase and compared them with those of the wild-type dehydrogenase. We sought rationales for changes in these rate constants in the structure of the mutant cocrystallized with the alternate substrate, 4-nitrobutyric acid. Substitution of aspartate for E370, the catalytic base, results in a 24% decrease in the rate constant for proton abstraction at C-2 of 3-thiaglutaryl-CoA as the distance between C-2 of the ligand and the closest carboxyl oxygen at residue 370 increases from 2.9 A to 3.1 A. The net rate constant for flavin reduction due to hydride transfer from C-3 of the natural substrate, which includes proton abstraction at C-2, to N5 of the flavin decreases by 81% due to the mutation, although the distance increases only by 0.7 A. The intensities of charge-transfer bands associated with the enolate of 3-thiaglutaryl-CoA, the reductive half-reaction (reduced flavin with oxidized form of substrate), and the dienolate following decarboxylation are considerably diminished. Structural investigation suggests that the increased distance and the change in angle of the S-C1(=O)-C2 plane of the substrate with the isoalloxazine substantially alter rates of the reductive and oxidative half-reactions. This change in active site geometry also changes the position of protonation of the four carbon dienolate intermediate to produce kinetically favorable product, vinylacetyl-CoA, which is further isomerized to the thermodynamically stable normal product, crotonyl-CoA.

Kinetic mechanism of glutaryl-CoA dehydrogenase.

Glutaryl-CoA dehydrogenase (GCD) is a homotetrameric enzyme containing one noncovalently bound FAD per monomer that oxidatively decarboxylates glutaryl-CoA to crotonyl-CoA and CO2. GCD belongs to the family of acyl-CoA dehydrogenases that are evolutionarily conserved in their sequence, structure, and function. However, there are differences in the kinetic mechanisms among the different acyl-CoA dehydrogenases. One of the unanswered aspects is that of the rate-determining step in the steady-state turnover of GCD. In the present investigation, the major rate-determining step is identified to be the release of crotonyl-CoA product because the chemical steps and reoxidation of reduced FAD are much faster than the turnover of the wild-type GCD. Other steps are only partially rate-determining. This conclusion is based on the transit times of the individual reactions occurring in the active site of GCD.

Protonation of crotonyl-CoA dienolate by human glutaryl-CoA dehydrogenase occurs by solvent-derived protons.

The protonation of crotonyl-CoA dienolate following decarboxylation of glutaconyl-CoA by glutaryl-CoA dehydrogenase was investigated. Although it is generally held that the active sites of acyl-CoA dehydrogenases are desolvated when substrate binds, recent evidence has established that water has access to the active site in these binary complexes of glutaryl-CoA dehydrogenase. The present investigation shows that the dehydrogenase catalyzes (a) a rapid exchange of C-4 methyl protons of crotonyl-CoA with bulk solvent and (b) protonation of crotonyl-CoA dienolate by solvent-derived protons under single turnover conditions. Both of the reactions require the catalytic base, Glu370. These findings indicate that decarboxylation proceeds via a dienolate intermediate. The involvement of water in catalysis by glutaryl-CoA dehydrogenase was previously unrecognized and is in conflict with a classically held intramolecular 1,3-prototropic shift for protonation of crotonyl-CoA dienolate.

Characterization of four variant forms of human propionyl-CoA carboxylase expressed in Escherichia coli.

Propionyl-CoA carboxylase (PCC) is a biotin-dependent mitochondrial enzyme that catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA. PCC consists of two heterologous subunits, alpha PCC and beta PCC, which are encoded by the nuclear PCCA and PCCB genes, respectively. Deficiency of PCC results in a metabolic disorder, propionic acidemia, which is sufficiently severe to cause neonatal death. We have purified three PCCs containing pathogenic mutations in the beta subunit (R165W, E168K, and R410W) and one PCCB polymorphism (A497V) to homogeneity to elucidate the potential structural and functional effects of these substitutions. We observed no significant difference in Km values for propionyl-CoA between wild-type and the variant enzymes, which indicated that these substitutions had no effect on the affinity of the enzyme for this substrate. Furthermore, the kinetic studies indicated that mutation R410W was not involved in propionyl-CoA binding in contrast to a previous report. The three mutant PCCs had half the catalytic efficiency of wild-type PCC as judged by the kcat/Km ratios. No significant differences have been observed in molecular mass or secondary structure among these enzymes. However, the variant PCCs were less thermostable than the wild-type. Following incubation at 47 degrees C, blue native-PAGE revealed a lower oligomeric form (alpha2beta2) in the three mutants not detectable in wild-type and the polymorphism. Interestingly, the lower oligomeric form was also observed in the corresponding crude Escherichia coli extracts. Our biochemical data and the structural analysis using a beta PCC homology model indicate that the pathogenic nature of these mutations is more likely to be due to a lack of assembly rather than disruption of catalysis. The strong favorable effect of the co-expressed chaperone proteins on PCC folding, assembly, and activity suggest that propionic acidemia may be amenable to chaperone therapy.

Crystal structures of human glutaryl-CoA dehydrogenase with and without an alternate substrate: structural bases of dehydrogenation and decarboxylation reactions.

Acyl-CoA dehydrogenases (ACDs) are a family of flavoenzymes that metabolize fatty acids and some amino acids. Of nine known ACDs, glutaryl-CoA dehydrogenase (GCD) is unique: in addition to the alpha,beta-dehydrogenation reaction, common to all ACDs, GCD catalyzes decarboxylation of glutaryl-CoA to produce CO(2) and crotonyl-CoA. Crystal structures of GCD and its complex with 4-nitrobutyryl-CoA have been determined to 2.1 and 2.6 A, respectively. The overall polypeptide folds are the same and similar to the structures of other family members. The active site of the unliganded structure is filled with water molecules that are displaced when enzyme binds the substrate. The structure strongly suggests that the mechanism of dehydrogenation is the same as in other ACDs. The substrate binds at the re side of the FAD ring. Glu370 abstracts the C2 pro-R proton, which is acidified by the polarization of the thiolester carbonyl oxygen through hydrogen bonding to the 2'-OH of FAD and the amide nitrogen of Glu370. The C3 pro-R proton is transferred to the N(5) atom of FAD. The structures indicate a plausible mechanism for the decarboxylation reaction. The carbonyl polarization initiates decarboxylation, and Arg94 stabilizes the transient crotonyl-CoA anion. Protonation of the crotonyl-CoA anion occurs by a 1,3-prototropic shift catalyzed by the conjugated acid of the general base, Glu370. A tight hydrogen-bonding network involving gamma-carboxylate of the enzyme-bound glutaconyl-CoA, with Tyr369, Glu87, Arg94, Ser95, and Thr170, optimizes orientation of the gamma-carboxylate for decarboxylation. Some pathogenic mutations are explained by the structure. The mutations affect protein folding, stability, and/or substrate binding, resulting in inefficient/inactive enzyme.

Mechanism-based inactivation of human glutaryl-CoA dehydrogenase by 2-pentynoyl-CoA: rationale for enhanced reactivity.

2-Pentynoyl-CoA inactivates glutaryl-CoA dehydrogenase at a rate that considerably exceeds the rates of inactivation of short chain and medium chain acyl-CoA dehydrogenases by this inhibitor and related 2-alkynoyl-CoAs. To determine the rate of inactivation by 2-pentynoyl-CoA, we investigated the inactivation in the presence of a non-oxidizable analog, 3-thiaglutaryl-CoA, which competes for the binding site. The enhanced rate of inactivation does not reflect an alteration in specificity for the acyl group, nor does it reflect the covalent modification of a residue other than the active site glutamate. In addition to determining the inactivation of catalytic activity a spectral intermediate was detected by stopped-flow spectrophotometry, and the rate constants of formation and decay of this charge transfer complex (lambdamax approximately 790 nm) were determined by global analysis. Although the rate-limiting step in the inactivation of the other acyl-CoA dehydrogenases can involve the abstraction of a proton at C-4, this is not the case with glutaryl-CoA dehydrogenase. Glutaryl-CoA dehydrogenase is also differentiated from other acyl-CoA dehydrogenases in that the catalytic base must access both C-2 and C-4 in the normal catalytic pathway. Access to C-4 is not obligatory for the other dehydrogenases. Analysis of the distance from the closest carboxylate oxygen of the glutamate base catalyst to C-4 of a bound acyl-CoA ligand for medium chain, short chain, and isovaleryl-CoA dehydrogenases suggests that the increased rate of inactivation reflects the carboxylate oxygen to ligand C-4 distance in the binary complexes. This distance for wild type glutaryl-CoA dehydrogenase is not known. Comparison of the rate constants of inactivation and formation of a spectral species between wild type glutaryl-CoA dehydrogenase and a E370D mutant are consistent with the idea that this distance in glutaryl-CoA dehydrogenase contributes to the enhanced rate of inactivation and the 1,3-prototropic shift catalyzed by the enzyme.

Alternate substrates of human glutaryl-CoA dehydrogenase: structure and reactivity of substrates, and identification of a novel 2-enoyl-CoA product.

The dehydrogenation reaction catalyzed by human glutaryl-CoA dehydrogenase was investigated using a series of alternate substrates. These substrates have various substituents at the gamma position in place of the carboxylate of the physiological substrate, glutaryl-CoA. The steady-state kinetic constants of the six alternate substrates and the extent of flavin reduction in the anaerobic half-reaction were determined. One of these substrates, 4-nitrobutyryl-CoA, was previously thought not to be a substrate of the dehydrogenase; however, the enzyme does oxidize this substrate analogue with a k(cat) that is less than 2% of that with glutaryl-CoA when ferrocenium hexafluorophosphate (FcPF(6)) is the electron acceptor. Anaerobic titration of the dehydrogenase with 4-nitrobutyryl-CoA showed no reduction of the flavin; but instead showed an increased absorbance in the 460 nm region suggesting deprotonation of the analogue to form the alpha-carbanion. Analysis of these data indicated a binding stoichiometry of about 1.0. Under aerobic conditions, a second absorption maximum is observed with lambda(max) = 366 nm. The generation of the latter chromophore is dependent on an electron acceptor, either O(2) or FcPF(6), and is greatly facilitated by the catalytic base Glu370. The 466 nm absorbing species remains enzyme-bound while the 366 nm absorbing species is present only in solution. The latter compound was identified as 4-nitronate-but-2-enoyl-CoA by mass spectrometry, (1)H NMR, and chemical analyses. Ionization of the enzymatic product, 4-nitro-but-2-enoyl-CoA, that yields the nitronate occurs in solution and not on the enzyme. The variation of k(cat) with the nature of the substituent suggests that the various substituents affect the free energy of activation, Delta G(++), for dehydrogenation. There is a good correlation between log(k(cat)) and F, the field effect parameter, of the gamma-substituent. No correlation was found between any other kinetic or equilibrium constants and the substituent parameters using quantitative structure-activity relationships (QSAR). 4-Nitrobutyryl-CoA is the extreme example with the strongly electron-withdrawing nitro group in the gamma position.