Why does ribonuclease irreversibly inactivate at high temperatures?

Zale, Stephen E.; Klibanov, Alexander M.

doi:10.1021/bi00367a014

Cited by 289 publications

(162 citation statements)

References 63 publications

Supporting

Mentioning

152

Contrasting

Order By: Relevance

“…The asterisk indicates the site of preferential hydrolysis. and 90 mC [71], and has been noted in lysozyme at pH 4 and 100 mC [70].…”

Section: Residues In Proteinsmentioning

confidence: 73%

“…However, during the 1980s it became clear that at 100 mC and above a number of irreversible reactions, including deamidation and peptide bond cleavage, occur in addition to denaturation [70,71]. Although it is difficult to place a theoretical upper temperature limit on the stability of proteins to denaturation, because of their universal applicability these irreversible degradative reactions would seem to limit protein stability to temperatures below, say, 120 mC.…”

Section: Resultsmentioning

confidence: 99%

“…The high energy of activation suggests that deamidation will occur at higher temperatures and explains the observation that, above 90 mC, deamidation is a major form of irreversible thermoinactivation in both lysozyme (pH 4, 6 and 8) [70] and ribonuclease (pH 6 and 8) [71].…”

Section: Figure 4 Hydrogen Bonding Of the Asn Side-chain Oxygenmentioning

confidence: 96%

“…However, in proteins a significant fraction of succinimide formation may originate from Asp residues. Indeed, this is the case in lysozyme [70] and ribonuclease [71], where significant irreversible thermo-inactivation was noted at pH 4 above 90 mC due to Asp hydrolysis. In calmodulin, succinimides are formed preferentially from Asp residues rather than Asn residues.…”

Section: Aspartate Residuesmentioning

confidence: 96%

“…Although conformational stability can be maintained for thermophilic proteins at high temperatures [2,3], irreversible degradative processes can lead to enzyme inactivation [70,71]. In contrast to denaturation, the irreversible processes of protein inactivation arise from changes in covalent bonding.…”

Section: Protein Degradation Above 80 MCmentioning

confidence: 99%

See 4 more Smart Citations

The denaturation and degradation of stable enzymes at high temperatures

Daniel

Dines

Petach³

1996

Biochemical Journal

271

175

View full text Add to dashboard Cite

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

show abstract

“…The asterisk indicates the site of preferential hydrolysis. and 90 mC [71], and has been noted in lysozyme at pH 4 and 100 mC [70].…”

Section: Residues In Proteinsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Figure 4 Hydrogen Bonding Of the Asn Side-chain Oxygenmentioning

confidence: 96%

Section: Aspartate Residuesmentioning

confidence: 96%

Section: Protein Degradation Above 80 MCmentioning

confidence: 99%

See 3 more Smart Citations

The denaturation and degradation of stable enzymes at high temperatures

Daniel

Dines

Petach³

1996

Biochemical Journal

271

175

View full text Add to dashboard Cite

show abstract

Influence of polymolecular events on inactivation behavior of xylose isomerase fromThermotoga neapolitana 5068

Hess

Kelly

1999

Biotechnol. Bioeng.

View full text Add to dashboard Cite

The inactivation behavior of the xylose isomerase from Thermotoga neapolitana (TN5068 XI) was examined for both the soluble and immobilized enzyme. Polymolecular events were involved in the deactivation of the soluble enzyme. Inactivation was biphasic at 95°C, pH 7.0 and 7.9, the second phase was concentration‐dependent. The enzyme was most stable at low enzyme concentrations, however, the second phase of inactivation was 3‐ to 30‐fold slower than the initial phase. Both phases of inactivation were more rapid at pH 7.9, relative to 7.0. Differential scanning calorimetry of the TN5068 XI revealed two distinct thermal transitions at 99° and 109°C. The relative magnitude of the second transition was dramatically reduced at pH 7.9 relative to pH 7.0. Approximately 24% and 11% activity were recoverable after the first transition at pH 7.0 and 7.9, respectively. When the TN5068 XI was immobilized by covalent attachment to glass beads, inactivation was monophasic with a rate corresponding to the initial phase of inactivation for the soluble enzyme. The immobilized enzyme inactivation rate corresponded closely to the rate of ammonia release, presumably from deamidation of labile asparagine and/or glutamine residues. A second, slower inactivation phase suggests the presence of an unfolding intermediate, which was not observed for the immobilized enzyme. The concentration dependence of the second phase of inactivation suggests that polymolecular events were involved. Formation of a reversible polymolecular aggregate capable of protecting the soluble enzyme from irreversible deactivation appears to be responsible for the second phase of inactivation seen for the soluble enzyme. Whether this characteristic is common to other hyperthermophilic enzymes remains to be seen. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 509–517, 1999.

show abstract

Effect of pH on rate of interfacial inactivation of serine proteases in aqueous-organic systems

Ross

Bell

Halling

2000

Biotechnol. Bioeng.

View full text Add to dashboard Cite

We studied the inactivation of trypsin and α‐ and β‐chymotrypsin by passage of droplets of tridecane though their aqueous solutions. The mechanism involves contact with the interface, because the loss of activity is proportional to the total area exposed. The rates of inactivation vary up to fivefold over the pH range 3 to 10. However, there is no clear maximum at the isoelectric point (pI) of each enzyme, where the amount of protein adsorbed is usually found to be highest. This is probably because, at the pI, there is also a minimum in structural alteration on adsorption. There may be a weak correlation with pH effects on foamability of the enzyme solutions, a parameter reported to reflect the “hardness” of different proteins, which controls their interfacial unfolding. The pH dependence of both inactivation and hardness cautions against attempts to correlate inactivation of different enzymes with a single value of a parameter such as adiabatic compressibility. There is no correlation between the effects of pH on interfacial inactivation and those reported in the literature on irreversible inactivation in concentrated urea or at high temperature. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 498–503, 2000.

show abstract

Why does ribonuclease irreversibly inactivate at high temperatures?

Cited by 289 publications

References 63 publications

The denaturation and degradation of stable enzymes at high temperatures

The denaturation and degradation of stable enzymes at high temperatures

Influence of polymolecular events on inactivation behavior of xylose isomerase fromThermotoga neapolitana 5068

Effect of pH on rate of interfacial inactivation of serine proteases in aqueous-organic systems

Contact Info

Product

Resources

About