The Unusual Kinetics of Rubredoxin Thermal Denaturation

Abstract: The thermal denaturation of the small iron protein, rubredoxin, has been previously shown to exhibit unusual kinetics (Elkins,1995) characterized by nearly constant rate of change and a reaction time independent of the starting amount of the protein. Recently, Wampler and Bradley (1997) have described a kinetic model that explains these data and a variety of other thermal denaturation data for various rubredoxins as well as other proteins. This presentation describes the features of the model and offers additional data that supports a working hypothesis where in rubredoxin thermal denaturation in the presense of oxygen leads to formation of an iron containing species that catalyzes further breakdown of the protein. In the absense of oxygen the kinetics are much slower and fit a more normal exponential decay model. Work is in progress to identify the products of this reaction (Neuhaus, 1997). This work was supported by NIH grants GM41482 and GM50736.


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Table of Contents

The Unusual Kinetics of Rubredoxin Thermal Denaturation

Outline of talk...

WHY KINETICS?

Characteristics of simple models

Some other examples:

Models, cont….

WHAT'S USUAL?

Lysozyme irreversible thermal denaturation at 100 degrees

this data...

Ferredoxin and Carbonic Anhydrase thermal denaturation.

WHY'S RD THERMAL DECAY UNUSUAL?

Chris Elkin’s data (1995) for Clostridium pasteurianum rubredoxin.

Unusual because...

With zero order...

Another look at the C. p. rubredoxin decays

A MODEL THAT FITS

Finding a model...

Finding a model...

Simple self-catalyzed model

Can you get Michaelis-Menten kinetics with an irreversible reaction?

...model can vary from exponential to very cooperative

More of Chris Elkin's data...

Therefore, Chris’s data shows characteristics of all aspects of the model mechanism.

Other thermal decays have similar characteristics: hydrogenase, glutamine synthetase, formyl tetrahydrofolate synthetase and alpha-glactosidase.

FITS TO THE MODEL

Spectra taken during aerobic Desulfovibrio vulgaris rubredoxin denaturation.

Proceedure...

Some fits to decay curves

Pyrococus furiosus rubredoxin decays are also fit.

Parameters from the fits

Some fits to the Clostridium pasteurianum rubredoxin decays

How do you interpret these fits...

Interpretation...

Interpretation...

CONCLUSIONS AND SPECULATION

review of fit models...

review of fit models...

Interpretation reiterated...

Other pertinent data...

Reality is always more complex.

Oxygen inhibits formation of 318 nm absorption band

Working Hypothesis:

Support from anaerobic study.

Support...

Other support:

Other support and suggested experiments

Author: J. E. Wampler

Home Page: this server

References Cited:

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