A correlation between protein thermostability and resistance to proteolysis

Daniel, Roy M.; Cowan, Don A.; Morgan, Hugh W.; Curran, Monique P

doi:10.1042/bj2070641

Cited by 139 publications

(67 citation statements)

References 18 publications

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“…By contrast, I3C NMR relaxation experiments have shown that no correlation exists between thermostability and mobility on the nanosecond time scale (Wuthrich et al, 1980). The increased resistance of thermophilic proteins to proteolysis (Daniel et al, 1982;Fontana, 1988) does not necessarily imply flexibility on a short time scale because global or subglobal unfolding may be required for proteolysis to occur. Such global or subglobal unfolding is probably irrelevant to enzyme catalysis.…”

Section: Discussionmentioning

confidence: 99%

Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin

1997

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Molecular dynamics simulations in solution are performed for a rubredoxin from the hyperthermophilic archaeon Pymcoccus furiosus (RdPf) and one from the mesophilic organism Desulfovibrio vulgaris (RdDv). The two proteins are simulated at four temperatures: 300 K, 373 K, 473 K (two sets), and 500 K; the various simulations extended from 200 ps to 1,020 ps. At room temperature, the two proteins are stable, remain close to the crystal structure, and exhibit similar dynamic behavior; the RMS residue fluctuations are slightly smaller in the hyperthermophilic protein. An analysis of the average energy contributions in the two proteins is made; the results suggest that the intraprotein energy stabilizes RdPf relative to RdDv. At 373 K, the mesophilic protein unfolds rapidly (it begins to unfold at 300 ps), whereas the hyperthermophilic does not unfold over the simulation of 600 ps. This is in accord with the expected stability of the two proteins. At 473 K, where both proteins are expected to be unstable, unfolding behavior is observed within 200 ps and the mesophilic protein unfolds faster than the hyperthermophilic one. At 500 K, both proteins unfold; the hyperthermophilic protein does so faster than the mesophilic protein. The unfolding behavior for the two proteins is found to be very similar. Although the exact order of events differs from one trajectory to another, both proteins unfold first by opening of the loop region to expose the hydrophobic core. This is followed by unzipping of the @-sheet. The results obtained in the simulation are discussed in terms of the factors involved in flexibility and thermostability.

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Section: Discussionmentioning

confidence: 99%

Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin

1997

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“…For example, the engineering of a 340-fold increase in stability at 100°C in a Bacillus stearothermophilus protease by eight site-specific mutations 34 was achieved without compromising catalytic activity, contradicting the dogma that molecular stability and activity are reciprocally related (through global conformational flexibility). Furthermore, there is good evidence for a general correlation between molecular stability to temperature and to other denaturing conditions 35,36 , suggesting common initiators and/or pathways of protein denaturation, whether induced by high temperature, extremes of pH, or the presence of organic solvents or detergents. The practical consequence of this observation is that the selection of a more thermostable enzyme variant (e.g., derived from a thermophilic source) or the engineering of enhanced thermostabil-ity may also result in increased molecular resistance to a range of deleterious conditions.…”

Section: Page |mentioning

confidence: 99%

The search for the ideal biocatalyst

2002

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While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.The past two decades have led to major advances in our understanding of the subtleties of protein structure-function interrelationships. Mechanisms of protein stability in aqueous and nonaqueous environments 1,2 , the links between conformational mobility, structural integrity and activity 3,4 , and the complexities of substrate specificity have all succumbed to the onslaught of advanced molecular methods, including crystallography 5-7 , site-specific mutagenesis, gene shuffling, and protein evolution [8][9][10][11] . Scientists are now in a position to visualize, if not design, catalytic systems that approach the functional "ideal".The "ideal" catalyst is typically considered by the biochemist in terms of turnover number (k cat ) or, for a given process, in terms of maximum specificity constant (A cat /KM). However, from a bioprocess viewpoint, each bioprocess is constrained by a set of conditions dictated by the specific properties of the substrates, products, and the bioconversion reaction. Thus, while for any given bioprocess it is clearly possible to specify a set of catalyst properties that would constitute the ideal for that process, only broad generalizations for ideality may otherwise be identified.In this review, we discuss molecular properties of enzymes from a bioprocess viewpoint. A paradigm for design based on the ideal process determining the desired features of the catalyst is presented. "Ideal" characteristics (generic and process-specific) and methodologies for seeking or engineering these are discussed. We then review the extent to which significant changes in protein functional properties have been achieved by application of these methods, together with issues that require more research and/or development. Ideal processesBioprocess engineers develop processes not only against fixed capital and operating expenditure, but also rapid, and often, very aggressive timelines. This is particularly the case for pharmaceutical products where patent expiry on the product demands a robust and scalable process with short development times 12 . Conventionally, once a synthetic route is fixed and the biocatalytic step defined, bioprocess design and operation are centered on the properties of the

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“…It is required so that enzymes have sufficient flexibility to perform their catalytic functions. An additional requirement for instability can be inferred from the finding that, irrespective of whether or not they are denatured, stable proteins are more resistant to proteolysis [65]. Excessively stable enzymes may therefore hinder the normal cellular turnover of enzymes.…”

Section: Inter-relationship Of Enzyme Stability and Activitymentioning

confidence: 99%

The denaturation and degradation of stable enzymes at high temperatures

Daniel

Dines

Petach³

1996

Biochemical Journal

277

175

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Now that enzymes are available that are stable above 100 mC it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in

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A correlation between protein thermostability and resistance to proteolysis

Cited by 139 publications

References 18 publications

Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin

Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin

The search for the ideal biocatalyst

The denaturation and degradation of stable enzymes at high temperatures

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