Progress 11/01/98 to 09/30/04
Outputs
Just prior to termination of my project, we tried to enhance the oxidase reactions of medium chain acyl-CoA dehydrogenase under diverse conditions of pH, buffer medium, and mutational states of the enzyme. We created several specific mutations in the vicinity of the active sites as well as peripheral regions. We also created double mutations which deepened the active sites of the enzyme (e.g., Glu-99-->Gly mutation) as well as created a cavity at the active site (Glu-376-->Gly/Ala). Unfortunately, none of these mutations promoted the oxidase reaction of the enzyme. We observed that the higher pH was more prone to promoting the oxidase reaction of the enzyme but at the expense of instability of the enzyme. No effect of buffer species was found on the oxidase versus dehydrogenase reactions of the enzyme.
Impacts
The experiments performed during this reporting period provided insight into the mechanistic aspect of Glu-376-->Gly/Thr-255-->Glu double mutant enzyme in eliciting unusual properties of the enzyme. These properties have been higher oxidase reaction, altered substrate specificity, and marked ionic strength sensitivity of the enzyme. However, these novel catalytic features of the enzyme do not appear to have immediate commercial impact.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
All our previous experimental data revealed that the enzyme-ligand complementarity is the hallmark to the origin of the oxidase reaction in medium chain acyl-CoA dehydrogenase. If the enzyme site cavity is optimally occupied by its substrate or product, the oxidase reaction is drastically suppressed. On the other hand, if the enzyme site cavity contains bulky groups or the cavities (due to site-specific mutations), the oxidase activity is considerably enhanced. However, since the magnitude of enhancement of the oxidase reaction was not adequate to be commercially viable (such as employing it as bactericidal agent), efforts have been made to enhance the oxidase, as well as other (commercially viable) activities of this enzyme by employing molecular biological techniques. In the above endeavors, we noted that Ghisla and collaborators switched the active site residues of medium chain acyl-CoA dehydrogenase such that the active site base (Glu) occupies the position
similar to that of long chain acyl-CoA dehydrogenase. Upon creating a double mutant (Glu-376-->Gly/Thr-255-->Glu), Ghisla's group noted that the mutant enzyme elicit both the oxidase reaction as well as alters the substrates specificity of the enzyme from medium to longer chain substrates. Toward understanding the mechanistic basis of the above mutation, we repeated their experiments and confirmed that the above mutation indeed enhances the oxidase reaction of the enzyme. However, to our further surprise, we realized that the above mutation also elicited the ionic strength sensitivity to the enzyme. The rate of the mutant enzyme catalyzed reaction was found to be inhibited (in a cooperative manner) as a function of the increasing concentrations of mono and divalent cations. The Hill coefficient and Ec50 values for the inhibition of the enzyme by different cations were found to vary in the range of 1.5-1.7 and 140-200 mM, respectively. The steady-state kinetic data revealed that
whereas the ionic strength had no effect on the kcat value of the enzyme, it cooperatively increased the Km value of the acyl-CoA substrates. The latter fact is further supported by the direct spectrophotometric titrations for the binding of chromophoric product, indoleacyloyl-CoA. A compilation of these data lead us to propose that ununsual properties of the Glu-376-->Gly/Thr-255-->Glu mutation originates from the hydrogen bonding between Glu-99 and Glu-255 within the active site of the enzyme.
Impacts
The experiments performed during this reporting period provided insight into the mechanistic aspect of Glu-376-->Gly/Thr-255-->Glu double mutant enzyme in eliciting unusual properties of the enzyme. These properties have been higher oxidase reaction, altered substrate specificity, and marked ionic strength sensitivity of the enzyme. However, these novel catalytic features of the enzyme does not appear to have immediate commercial impact.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
During this reporting period, we attempted to answer as to why the oxidase activity of medium chain acyl-CoA dehydrogenase was suppressed and that of acyl-CoA oxidase is so pronounced. A casual comparison of the DNA sequence between these two enzymes revealed that the acyl-CoA oxidase was considerably larger than the dehydrogenase. The molecular model building studies using Homology lead to the suggestion that the extraneous protein fragment of the oxidase (about 20 KD) was not involved in forming the active site of the enzyme. Besides, acyl-CoA oxidase could predominate in a higher oligomeric state. We discovered that the enzyme activity of acyl-CoA oxidase decreased as the extent of oligomerization increased. For example, glycerol was found to shift the equilibrium between the dimeric form of the enzyme to the tetrameric form, with concomitant impairment of the enzyme activity. Such an equilibrium transition was not observed with medium chain acyl-CoA dehydrogenase.
We are currently investigating whether the functional difference in these enzymes is dictated by their structural differences.
Impacts
The comparative studies between acyl-CoA dehydrogenase and acyl-CoA oxidase have been important for understanding the control of the oxidase reaction, which has potential to serve as the food preservative.
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
In order to understand the molecular basis for the origin of the oxidase reaction in acyl-CoA dehydrogenase, we purified and characterized acyl-CoA oxidase from yeast, Yarrowia lipolytica, in collaboration with Dr. Jan Marc Nicaud's group in France, as well investigated the influence of Glu-376 to Gln mutation of the dehydrogenase. The yeast acyl-CoA oxidase (isoenzyme-3, abbreviated as Aox3p) has been cloned in E. coli with a 6 His tag sequence at 5'-end of the coding region. The His-tag containing protein was purified to homogeneity via the Ni-Nta and FPLC-TMAE column chromatographic techniques. The purified enzyme exhibited the oxidase reaction with a wide variety of substrates, including the aliphatic acyl-CoA substrates of chain lengths C6 to C14 as well as heterocyclic/aromatic ring substituted substrate such as furyl-propionyl-CoA. Based on the kinetic data, the efficiency of the enzyme for different substrates was found to be in the following order,
decanoyl-CoA,myristyl-CoA,hexanoyl-CoA, furyl-propionyl-CoA. While investigating the substrate specificity, it was noted that phenol, which is used in the coupled assay system for monitoring the enzyme activity, serves both as activator (at low concentration) and inhibitor (at high concentration) of the enzyme. We are currently in the process of investigating the influence of phenol and another effector, glycerol, on the oligomeric states of the enzyme. Aside from acyl-CoA oxidase, we continued our studies on the influence of the electrostatic field of the enzyme and ligand phases on formation of the enzyme-ligand complexes, the process intimately linked to the origin of the oxidase reaction. We investigated the influence of Glu-376 to Gln mutation on the thermodynamic properties for the binding of 2-azaoctanoyl-CoA and octenoyl-CoA to the enzyme. The microcalorimetric data revealed that unlike the wild-type enzyme, the enthalpy and heat capacity changes for the binding of the above
ligands to mutant enzyme were the same. A detailed analysis of experimental data led to the suggestion that the origin of the above thermodynamic features lies in solvent reorganization and water mediated electrostatic interaction between ligands and enzyme site groups, and such interactions were argued to be intrinsic to the molecular basis of enzyme-ligand complementarity.
Impacts
Our recent approach to undertake comparative studies utilizing the recombinant form of acyl-CoA oxidase from yeast is intended to throw light on the molecular basis for the origin of the oxidase reaction in acyl-CoA dehydrogenase. Since the electrostatic field of the enzyme and ligand site phases appear to be control the opening and closing of the enzyme sites during the course of ligand binding and catalysis, the features which are responsible for the oxidase reaction, the site specific mutation studies involving Glu-376 to Gln enzyme were intended to throw light on the mechanistic feature of the enzyme.
Publications
- Luo, Y.S., Wang, H.J., Gopalan, K. V., Srivastava, D. K., Nicaud, J.M., Chardot, T.(2000)Purification and characterization of the recombinant form of acyl-CoA oxidase 3 from the yeast Yarrowia lipolytica. Arch. Biochem. Biopphys. 384, 1-8.
- Peterson, K. M., Gopalan, K. V., Nandy, A., and Srivastava, D. K. (2001) Influence of Glu-376 to Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase. Protein Sci., 10, 1822-1834.
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Progress 10/01/99 to 09/30/00
Outputs
During this period, we attempted to understand the molecular basis of the enzyme-ligand complementarity, involving medium-chain acyl-CoA dehydrogenase. Such studies were prompted by our preliminary observation that the cavity creating mutations in the enzyme enhanced its "oxidase" reaction. The latter reaction has been essential for producing hydrated fatty acid products for industrial applications. In this endeavor, we compared the sequence of events involved during the course of binding of octenoyl-CoA (the reaction product of the enzyme) and 2-aza-octanoyl-CoA (the alpha-CH2 to NH substituted octanoyl-CoA) to the wild-type medium-chain acyl-CoA dehydrogenase. The experimental data revealed that both these ligands bound to the enzyme via a two-step mechanism, albeit with marked difference in their intrinsic binding affinites. Whereas octanoyl-CoA was preferentially stabilized within the enzyme-ligand collision complex, 2-azaoctanoyl-CoA favored the enzyme-ligand
isomerization equilibrium. Interestingly, the overall binding constants for both these ligands to the enzyme site were essentially the same. A detailed analysis of the experimental data revealed that the electrostatic contributions, imparted by the NH-group of 2-azaoctanoyl-CoA, were essential for enthalpically stabilizing the ground and transition states during the course of the enzyme-ligand isomerization process. We further investigated the influence of Glu-376 to Asp mutation, which showed evidence of enhancing the oxidase activity of the enzyme, on accommodating 2-azaoctanoyl-CoA and 2-azadithiooctanoyl-CoA ligands. The difference in the molecular volume between the above ligands (11 cubic angstrom) was deduced to be similar to the volume of the cavity created via the Glu-376 to Asp mutation. A comparative account of the ligand binding data revealed that the above mutation favorably accommodated the bulkier ligand at its site, suggesting that medium-chain acyl-CoA dehydrogenase
exhibits the potential of adjusting its internal cavities for accommodating bulkier ligands. This feature of the enzyme provides insights into the variation of the oxidase reaction as a function of the substrate type as well as the site-specific mutations.
Impacts
These studies have been intended to throw light on the molecular basis of the enzyme-ligand complementarity. We have investigated the above features by changing the ligand structure as well as by creating an active site cavity via Glu-376 to Asp mutation in medium-chain acyl-CoA dehydrogenase. The potential of the enzyme to accomodate differently substituted ligands has been the hallmark of understanding the activation and inhibition of the "oxidase" reaction as a function of the substrate type as well as the site-specific mutations.
Publications
- Peterson, K. M., Gopalan, K. V., and Srivastava, D. K. (2000)"Influence of alpha-CH2 to NH substitution in C8-CoA on the kinetics of association and dissociation of ligands with medium chain acyl-CoA dehydrogenase" Biochemistry 41, 12659-12670.
- Peterson, K. M., and Srivastava, D. K. (2000) "Energetic consequences of accomodating a bulkier ligand at the active site of medium chain acyl-CoA dehydrogenase by creating a complementary enzyme site cavity" Biochemistry 39, 12678-12687.
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Progress 10/01/98 to 09/30/99
Outputs
During this reporting period, we focused our attention toward understanding the molecular basis of inhibition of recombinant acyl-CoA dehydrogenase by phenolic compounds. We tested differently substituted (Cl, NO2, OH) phenolic compounds on the oxidase and dehydrogenase activities of the enzyme. All phenolic compounds were found to activate the oxidase reaction of the enzyme at low (<1 mM) concentrations, and inhibit both oxidase and dehydrogenase reactions at high (>1 mM) concentrations. Whereas the activation pattern of the enzyme is conformed to the Michaelis-dependence on the concentrations of phenolic compounds, the inhibition patterns have been found to be sigmoidal in nature. The inhibition data were best fitted by the Hill equation, with varied magnitudes (depending upon the inhibitor type) of the Hill coefficient and Ec50 (the concentration of inhibitor required to achieve 50% inhibition) values. However, to our surprise, the magnitude of the Hill coefficient
was not only dependent upon the nature of the phenolic compound, but also upon the type of the enzyme's substrate, utilized during the activity measurement. For example, with indolepropionyl-CoA as a chromogenic substrate, the magnitudes of the Hill coefficients of phenolic compounds were 2-5 fold lower than those obtained with octanoyl-CoA as a physiological substrate. Although at this time, we do not understand the mechanistic origin of the above disparity, we conjecture that the origin of the above variation lies in the prevalence of multiple binding sites of phenolic compounds at the enzyme surface. However, on consideration that only a few of such sites would be involved in the inhibition of the enzyme, we analyzed the (sigmoidal) inhibitory profiles for the dichlorophenol and salicylic acid-dependent reactions according to our previously established graphical method (Wang,Z.X. and Srivastava,D.K., Anal. Biochem. 216,15-26,1994). Such analyses revealed that the complete
inhibition of the enzyme is accomplished via binding of two molecules of phenolic compounds per molecule of the enzyme. At this time, it is unclear as to whether or not the above stoichiometry pertains to the binding of two molecules of phenolic compounds per enzyme subunit or to the overall tetramer. We are currently evaluating the above possibilities via the isothermal microcalorimetric titration method.
Impacts
We are trying to explore the possibility of determining small quantities of phenolic compounds, present as contaminants in drinking water, via an enzymatic method. We have identified, medium chain acyl-CoA dehydrogenase as a potential enzyme to accomplish the above goals. During this reporting period, we have attempted to understand the functional aspects of the enzymatic reactions and the overall stoichiometry of the enzyme-inhibitor complex.
Publications
- No publications reported this period
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