scholarly journals Purification and properties of erythro-β-hydroxyasparate dehydratase from Micrococcus denitrificans

1965 ◽  
Vol 97 (2) ◽  
pp. 547-554 ◽  
Author(s):  
RG Gibbs ◽  
JG Morris
Keyword(s):  
Pyridoxal Phosphate ◽  
Competitive Inhibitor ◽  
The Novel ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Bivalent Cations ◽  
Purification And Properties ◽  
Key Enzyme ◽  
Aldolase Activity ◽  
No Activation

1. The novel enzyme, erythro-beta-hydroxyaspartate dehydratase, a key enzyme of the beta-hydroxyaspartate pathway (Kornberg & Morris, 1963, 1965), has been purified 30-fold from extracts of glycollate-grown Micrococcus denitrificans. The purified preparation was devoid of erythro-beta-hydroxyaspartate-aldolase activity, and free from enzymes that act on oxaloacetate. 2. Properties of the purified dehydratase were studied by direct assay of the enzymic formation of oxaloacetate and ammonia from added erythro-beta-hydroxyaspartate. 3. The enzyme was highly substrate-specific, utilizing only the l-isomer of erythro-beta-hydroxyaspartate (K(m), 0.43mm, and V(max.), 99mumoles of oxaloacetate formed/min./mg. of protein at pH9.15 and 30 degrees). Of many compounds tested, only maleate was a competitive inhibitor (K(i), 32mm at pH7.6). 4. The optimum pH for activity was about 9.5. The K(m) varied with pH, showing a marked optimum at pH7.8. The V(max.) also varied with pH in a manner suggesting the presence in the enzyme-substrate complex of a dissociable group of pK‣(a) about 8.5. 5. Carbonyl reagents were inhibitory, but of three thiol reagents tested only p-chloromercuribenzoate was inhibitory. 6. A partially resolved preparation of the enzyme was activated four-fold by the addition of pyridoxal phosphate and thereby restored to half activity. 7. EDTA (0.1mm) was almost completely inhibitory, activity being restored by bivalent cations (Mg(2+), Ca(2+) and Mn(2+)); no activation by univalent cations was observed. 8. The findings are discussed in the light of reported properties of related hydroxyamino acid dehydratases.

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

scholarly journals Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex

1977 ◽  
Vol 167 (2) ◽  
pp. 361-366 ◽  
Author(s):  
E Grazi ◽  
M Blanzieri
Keyword(s):  
Competitive Inhibitor ◽  
Dihydroxyacetone Phosphate ◽  
Rabbit Muscle ◽  
Keto Form ◽  
First Order ◽  
Phosphate Complex ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Dead End ◽  
Fructose 1,6 Bisphosphate

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.

A KINETIC STUDY OF THE DEAMINATION OF SOME ADENOSINE ANALOGUES: SUBSTRATE SPECIFICITY OF ADENOSINE DEAMINASE

1966 ◽  
Vol 44 (3) ◽  
pp. 331-337 ◽  
Author(s):  
J. Lyndal York ◽  
G. A. LePage
Keyword(s):  
Substrate Specificity ◽  
Kinetic Study ◽  
Adenosine Deaminase ◽  
Competitive Inhibitor ◽  
Kinetic Constants ◽  
Glycosidic Linkage ◽  
Adenosine Analogues ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Marked Dependence

The kinetic constants Km and Vmax were determined for the deamination by adenosine deaminase of a series of analogues of adenosine containing "fraudulent" sugars. The configuration of the 2′-hydroxyl was found to be important for the binding of enzyme and substrate. The largest effect of changes in sugar structure was on the rate of breakdown of the enzyme–substrate complex to form products, i.e. Vmax. The nature of the configuration in the 3′-position was not important if the 2′-hydroxyl was trans to the glycosidic linkage; however, if the steric arrangement of the 2′-hydroxyl was cis to the glycosidic linkage, then Vmax showed a marked dependence on the nature of the 3′-substituent and its configuration. For instance, Vmax values were for arabinosyl adenine < 3′-deoxyarabinosyl adenine <lyxosyl adenine. 6-N-methyladenosine was found to be a competitive inhibitor of adenosine deaminase, with a Ki of 2 × 10−6M.

Enzyme–substrate complex structures of CYP154C5 shed light on its mode of highly selective steroid hydroxylation

2014 ◽  
Vol 70 (11) ◽  
pp. 2875-2889 ◽  
Author(s):  
Konrad Herzog ◽  
Paula Bracco ◽  
Akira Onoda ◽  
Takashi Hayashi ◽  
Kurt Hoffmann ◽  
...  
Keyword(s):  
Polar Regions ◽  
Complex Structures ◽  
P450 Monooxygenase ◽  
Hormone Synthesis ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Structural Differences ◽  
Key Enzyme ◽  
Steroid Binding ◽  
Shed Light

CYP154C5 fromNocardia farcinicais a bacterial cytochrome P450 monooxygenase active on steroid molecules. The enzyme has recently been shown to exhibit exclusive regioselectivity and stereoselectivity in the conversion of various pregnans and androstans, yielding 16α-hydroxylated steroid products. This makes the enzyme an attractive candidate for industrial application in steroid hormone synthesis. Here, crystal structures of CYP154C5 in complex with four different steroid molecules were solved at resolutions of up to 1.9 Å. These are the first reported P450 structures from the CYP154 family in complex with a substrate. The active site of CYP154C5 forms a flattened hydrophobic channel with two opposing polar regions, perfectly resembling the size and polarity distribution of the steroids and thus resulting in highly specific steroid binding withKdvalues in the range 10–100 nM. Key enzyme–substrate interactions were identified that accounted for the exclusive regioselectivity and stereoselectivity of the enzyme. Additionally, comparison of the four CYP154C5–steroid structures revealed distinct structural differences, explaining the observed variations in kinetic data obtained for this P450 with the steroids pregnenolone, dehydroepiandrosterone, progesterone, androstenedione, testosterone and nandrolone. This will facilitate the generation of variants with improved activity or altered selectivity in the future by means of protein engineering.

scholarly journals The pyridoxal-binding site in pyridoxamine-pyruvate transaminase

1978 ◽  
Vol 169 (2) ◽  
pp. 429-432 ◽  
Author(s):  
J Hodsdon ◽  
H Kolb ◽  
E E Snell ◽  
R D Cole
Keyword(s):  
Binding Site ◽  
Pyridoxal Phosphate ◽  
Tryptic Digestion ◽  
Substrate Complex ◽  
Chromatography Analysis ◽  
Enzyme Substrate ◽  
Lysine Residues

The enzyme-substrate complex formed between pyridoxamine-pyruvate transaminase (EC 2.6.1.30) and pyridoxal was reduced with NaBH4. After carboxymethylation and tryptic digestion, pyridoxyl-lysine-containing peptides were isolated by a combination of Sephadex and Dowex 50 chromatography. Analysis of these peptides shows the structure around the pyridoxal-binding lysine residues to be Ala-Asp-Ile-Tyr-Val-Thr-Gly-Pro-Asx-Lys(Pxy)-Cys-Leu(Pro2, Gly2, Ala2, Met)(Thr, Leu2)Gly-Val-Ser-Glu-Arg. This structure differs from those found for the corresponding peptides from pyridoxal phosphate-dependent enzymes.

Transient Formation of a Complex Between α-Chymotrypsin, Proflavin, and Tosylarginine Methyl Ester (TAME)

1972 ◽  
Vol 50 (3) ◽  
pp. 257-260 ◽  
Author(s):  
George H. Czerlinski ◽  
Catherine Odell
Keyword(s):  
Methyl Ester ◽  
Ternary Complex ◽  
Competitive Inhibitor ◽  
Relaxation Processes ◽  
Relaxation Time Constant ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Fast Relaxation ◽  
Chemical Relaxation ◽  
Relaxation Experiments

Chemical relaxation experiments were conducted on the reaction of α-chymotrypsin, with the competitive inhibitor proflavin and the substrate analogue TAME (tosylarginine methyl ester) in phosphate buffer, pH 6.7, observing transmission changes at 465 mμ. Two chemical relaxation processes were observed with the slow one attributed to a monomolecular interconversion of the enzyme–substrate complex. The concentration dependence of the reciprocal fast relaxation time constant only agrees with the equations derived for the involvement of a labile ternary complex between enzyme, substrate, and inhibitor (as simplest model).

scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

scholarly journals Conformational Analysis of the Enzyme-Substrate Complex in the Dehydrogenation of Sterols by Tetrahymena pyriformis

1971 ◽  
Vol 246 (3) ◽  
pp. 561-568 ◽  
Author(s):  
William R. Nes ◽  
P.A. Govinda Malya ◽  
Frank B. Mallory ◽  
Karen A. Ferguson ◽  
Josephine R. Landrey ◽  
...  
Keyword(s):  
Conformational Analysis ◽  
Tetrahymena Pyriformis ◽  
Substrate Complex ◽  
Enzyme Substrate

scholarly journals N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

2021 ◽  
Vol 49 (5) ◽  
pp. 2684-2699
Author(s):  
Ka-Weng Ieong ◽  
Gabriele Indrisiunaite ◽  
Arjun Prabhakar ◽  
Joseph D Puglisi ◽  
Måns Ehrenberg
Keyword(s):  
Single Molecule ◽  
Stop Codon ◽  
Product Formation ◽  
Release Factors ◽  
Substrate Complex ◽  
Peptidyl Transfer ◽  
Enzyme Substrate ◽  
Peptide Release ◽  
Accuracy Ratio ◽  
Codon Reading

Abstract We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

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