scholarly journals The significance of denaturant titrations of protein stability: a comparison of rat and baker's yeast cytochrome c and their site-directed asparagine-52-to-isoleucine mutants

1994 ◽  
Vol 299 (2) ◽  
pp. 347-350 ◽  
Author(s):  
T I Koshy ◽  
T L Luntz ◽  
B Plotkin ◽  
A Schejter ◽  
E Margoliash
Keyword(s):  
Cytochrome C ◽  
Baker’S Yeast ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Baker's Yeast ◽  
Guanidinium Chloride ◽  
Mutant Proteins ◽  
Cytochromes C ◽  
Structural Differences ◽  
The Stability

The residue asparagine-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was mutated to isoleucine by site-directed mutagenesis, and the unfolding of the wild-type and mutant proteins in urea or guanidinium chloride solutions was studied. Whereas the yeast mutant cytochrome unfolded in 4-7 M urea with a rate constant (k) of 1.7 x 10(-2) s-1, the rat mutant protein unfolded with k = 5.0 x 10(-2) s-1, followed by a slow partial refolding with k = 5.0 x 10(-4) s-1. Denaturant titrations indicated that the mutation increased the stability of the yeast cytochrome by 6.3 kJ (1.5 kcal)/mol, while it decreased that of the rat protein by 11.7 kJ (2.8 kcal)/mol. These results probably reflect structural differences between yeast iso-1 and vertebrate cytochromes c in the vicinity of the Asn-52 side chain.

scholarly journals Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c

1994 ◽  
Vol 302 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A Schejter ◽  
T I Koshy ◽  
T L Luntz ◽  
R Sanishvili ◽  
I Vig ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
General Increase ◽  
Reduction Potentials ◽  
Cytochromes C ◽  
In The Wild ◽  
Order Of Magnitude ◽  
The Stability ◽  
Haem Iron

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

Studies on cytochrome c. X. Synthesis ofN?-benzyloxycarbonyl-[Thr107]-dotetracontapeptide (sequence 67-108) of baker's yeast iso-1-cytochrome c

Biopolymers ◽  
1975 ◽  
Vol 14 (10) ◽  
pp. 2061-2074 ◽  
Author(s):  
Luis Moroder ◽  
Bruno Filippi ◽  
Gianfranco Borin ◽  
Fernando Marchiori
Keyword(s):  
Cytochrome C ◽  
Baker’S Yeast ◽  
Baker's Yeast

Studies on cytochrome c. Part IV. Synthesis of the protected dodecapeptide (sequence 45-56) of Baker's yeast iso-1-cytochrome c

Biopolymers ◽  
1973 ◽  
Vol 12 (3) ◽  
pp. 521-534 ◽  
Author(s):  
Gianfranco Borin ◽  
Luis Moroder ◽  
Fernando Marchiori ◽  
Ernesto Scoffone
Keyword(s):  
Cytochrome C ◽  
Baker’S Yeast ◽  
Baker's Yeast

Reactive Amino Groups in Baker's Yeast Cytochrome c

1966 ◽  
Vol 60 (6) ◽  
pp. 682-690
Author(s):  
CHIAKI SATO ◽  
KOITI TITANI ◽  
KOZO NARITA
Keyword(s):  
Cytochrome C ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Amino Groups

scholarly journals The unfolding and refolding of glutamate dehydrogenases from bovine liver, baker's yeast and Clostridium symbosium

1988 ◽  
Vol 251 (1) ◽  
pp. 135-139 ◽  
Author(s):  
S M West ◽  
N C Price
Keyword(s):  
Enzyme Activity ◽  
Light Scattering ◽  
Liver Enzyme ◽  
Structural Changes ◽  
Bovine Liver ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Guanidinium Chloride ◽  
Reversible Loss ◽  
Parallel Behaviour

The unfolding behaviour of the hexameric glutamate dehydrogenases from bovine liver, Clostridium symbosium and baker's yeast in solutions of guanidinium chloride (GdnHCl) was studied. Changes in Mr studied by light-scattering indicate that, in each case, the hexamer dissociates to form trimers, which then dissociate to monomers at higher concentrations of GdnHCl. Dissociation to trimers is accompanied by a reversible loss of enzyme activity, but no gross structural changes can be detected by fluorescence or c.d. Dissociation to monomers is accompanied by large structural changes, and the loss of activity cannot be reversed by dilution. The parallel behaviour of all three enzymes shows that the previously noted inability of the isolated subunits of the bovine liver enzyme to refold [Bell & Bell (1984) Biochem. J. 217, 327-330] is not a result of any modification of the enzyme as a result of import into mitochondria, since the C. symbosium and baker's-yeast enzymes do not undergo any such post-translational translocation.

N- and C-Terminal Residues in Baker's Yeast Cytochrome c

1964 ◽  
Vol 56 (3) ◽  
pp. 222-229 ◽  
Author(s):  
YOSHIHITO YAOI ◽  
KOITI TITANI ◽  
KOZO NARITA
Keyword(s):  
Cytochrome C ◽  
Baker’S Yeast ◽  
Baker's Yeast

scholarly journals Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Canfang Niu ◽  
Peilong Yang ◽  
Huiying Luo ◽  
Huoqing Huang ◽  
Yaru Wang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Side Chains ◽  
Cleavage Sites ◽  
Corn Meal ◽  
Feed Enzymes ◽  
The Stability ◽  
Phytate Content

Abstract Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. We constructed mutants with different side chain structures at these sites using site-directed mutagenesis and produced all enzymes in Escherichia coli for catalytic and biochemical characterization. The substitutions E230G/A/P/R/S/T/D, L162G/A/V, L99A, L99A/L162G, and L99A/L162G/E230G improved the pepsin resistance. Moreover, E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3–2.4-fold, improved their stability at 60 °C and pH 1.0–2.0 and alleviated inhibition by metal ions. In addition, E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. This study proposes an efficient approach to improve enzyme performance in monogastric animals fed feed with a high phytate content.

Studies on cytochrome c. Part II. Synthesis of the protected heptapeptide (sequence 17-23) of Baker's yeast iso-1-cytochrome c

Biopolymers ◽  
1973 ◽  
Vol 12 (3) ◽  
pp. 493-505 ◽  
Author(s):  
Luis Moroder ◽  
Fernando Marchiori ◽  
Gianfranco Borin ◽  
Ernesto Scoffone
Keyword(s):  
Cytochrome C ◽  
Baker’S Yeast ◽  
Baker's Yeast
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