The denaturation and degradation of stable enzymes at high temperatures

Daniel, Roy M.; Dines, Mark; Petach, Helen H.

doi:10.1042/bj3170001

Cited by 266 publications

(189 citation statements)

References 103 publications

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“…According to Daniel et al (1996), biphasic thermal inactivation curves like that observed in Figure 5, can reflect the ability of the enzyme to exist in more than one stable conformational state. Since β-gal III was almost completely inactivated upon pretreatment at 60°C for 40 minutes, and β-gal I and II enzymes resisted until 80 minutes at the same pretreatment, it is possible that the β-galactosidase derived from cell wall (β-gal III) is more sensitive to thermal inactivation than the cytosolic counterparts (β-gal I and II).…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of cytosolic and cell wall β-galactosidases from Vigna unguiculata stems

Sudério

Barbosa²,

Gomes-Filho³

et al. 2011

Braz. J. Plant Physiol.

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Three β-galactosidase isoforms, β-gal I and β-gal II (cytosolic) and β-gal III (cell wall-associated), were isolated from stems of Vigna unguiculata (L.) Walp. cv. Pitiúba seedlings. Purification consisted of ammonia sulfate fractionation followed by chromatography in DEAE-Sephadex and Lactosyl-Sepharose columns. The two cytosolic isoforms showed the same chromatography pattern, which differed from that of β-gal III. Electrophoresis revealed a single band of protein for β-gal II and β-gal III which also expressed β-galactosidase activity in gel. The apparent molecular mass of the β-gal I, II and III was 89, 146 and 124 kDa, respectively. The three isoforms revealed the same optimal pH (4.0) and the same optimal assay temperature (55°C) for enzyme activity. The three isoforms were stable at temperatures up to 50°C, and incubation with glucose and galactose expanded their thermal stability as well as inhibited their activities. Galactose was the most effective in promoting these effects and β-gal I and II were competitively inhibited by this sugar. Kinetic analysis using β-PNPG as substrate, revealed KM of 1.69, 1.76 and 1.43 for β-gal I, β-gal II and β-gal III, respectively. The β-gal I was able to hydrolyze all synthetic substrates tested, whereas β-gal II exhibited only β-fucosidase and a-arabinosidase activities, and β-gal III was limited to the a-galactosidase, β-fucosidase and a-arabinosidase activities. These results are consistent with three distinct β-galactosidases exhibiting quite similar kinetic features, but endowed with different functional specificities probably related to their specific roles in the plant cell physiology.

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of cytosolic and cell wall β-galactosidases from Vigna unguiculata stems

Sudério

Barbosa²,

Gomes-Filho³

et al. 2011

Braz. J. Plant Physiol.

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“…The present results are in perfect agreement with these observations, the first calcium to be released being the one bound in the Ca3 site, which, however, as the above results also indicate, is not predominant in determining stability. Further studies of the contribution of calcium-ion binding to stability, as well as attempts to use the information gained in the present study to design TLP variants with characteristics resembling those of enzymes isolated from hyperthermophilic archea [57] are currently in progress in our laboratory.…”

Section: Mutations In the N-terminal Turn Of The 68 To 87 A-helixmentioning

confidence: 99%

Mutational Analysis of a Surface Area that is Critical for the Thermal Stability of Thermolysin‐Like Proteases

Veltman

Vriend²,

Hardy

et al. 1997

European Journal of Biochemistry

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Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55 -69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp.59). In the case of the AspS7+Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.Keywords: thermal stability; thermolysin; autolysis ; calcium binding; unfolding pathway.Bacillus thermolysin-li ke proteases (TLP) are metallo-endopeptidases consisting of 300-3 19 residues. These enzymes contain one zinc ion that is essential for catalysis and they bind a varying number of calcium ions for which it is known that they contribute to stability [I -21. The amino acid sequences of many TLP are known and the crystal structures of thermolysin (the TLP of Bacillus thermoproteolyticus; [3,41) and TLP-cer (the TLP of Bacillus cereus; [S]) have been determined. TLP consist of an N-terminal domain (residues 1 -154; thermolysin numbering) characterized by a predominance of P-pleated sheet and a C-terminal domain (residues 155-31 6) that is mainly a-helical [4, 51. TLP differ in thermal stability [6] and the structural determinants of these differences have been analyzed by several sitedirected mutagenesis studies [7-111. At elevated temperatures TLP are irreversibly inactivated as a result of autolysis. Considering the broad specificity of TLP [ 12, 131, conformational Enzyme. Thermolysin (EC 3.4.24.27).proteolytic attack [14]. It has been shown that local unfolding processes that render the protein susceptible towards autolysis are the rate-limiting steps in thermal inactivation [6,[15][16]. It has been proposed that these thermally induced local unfolding processes mainly involve surface located regions of the protein 16, 9, 141. Accordingly, it has been shown that the changes in the stability of TLP-ste is most easily effected by mutation of surface-located residues that are clustered in one particular region (residues 55-69) of the protein [9, 111 (and see Figs 1 and 2). The difference in stability between thermolysin (tso = 86.5 "C ; see below for definition of tS0) and the TLP of B. stemothermophilus CU21 (TLP-ste; t50 = 73.4"C) ...

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“…Deamidation of Asn residues is not a likely event to occur at 40-45 mC, and phosphate ions have been reported to inhibit Asn deamidation in lysozyme [40]. However, Asn-133 is part of a Gly-rich hexapeptide (Gly-Asn-Gly-Gly-Leu-Gly) of the sequence, and it has been shown that conformational flexibility, eventually brought about by glycines, can facilitate deamidation [41]. Deamidation with concomitant release of NH $ does not occur to a measurable extent at 45 mC, ruling out its contribution to inactivation of MalP.…”

Section: Stability and Activity Of Wild-type Malp And The Asn-133 Alamentioning

confidence: 99%

Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

Griessler

D’Auria

Schinzel

et al. 2000

Biochemical Journal

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Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each $ 90-kDa subunit contains activesite pyridoxal 5h-phosphate. To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133 Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at $ 45 mC. They are manifested by slight increases in unordered structure and "H\#H exchange, and reflect reversible inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at $ 45 mC without prior release into solution of pyridoxal 5h-phosphate. It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme. Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP

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The denaturation and degradation of stable enzymes at high temperatures

Cited by 266 publications

References 103 publications

Purification and characterization of cytosolic and cell wall β-galactosidases from Vigna unguiculata stems

Purification and characterization of cytosolic and cell wall β-galactosidases from Vigna unguiculata stems

Mutational Analysis of a Surface Area that is Critical for the Thermal Stability of Thermolysin‐Like Proteases

Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

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