scholarly journals Studies of the esterase activity of cytosolic aldehyde dehydrogenase with resorufin acetate as substrate

1997 ◽  
Vol 322 (3) ◽  
pp. 701-708 ◽  
Author(s):  
Trevor M. KITSON ◽  
Kathryn E. KITSON
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Point Of View ◽  
Ph Profile ◽  
Rate Limiting Step ◽  
The Common ◽  
Aldehyde Oxidation ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Very High ◽  
Putative Catalytic Residue

Resorufin acetate is a very good substrate for sheep liver cytosolic aldehyde dehydrogenase, both from the point of view of practical spectrophotometry and in terms of information provided about the nature of the catalysis shown by this enzyme. p-Nitrophenyl (PNP) acetate competes against resorufin acetate for the enzyme's active site (although relatively weakly as the latter substrate has the lower Michaelis constant), but acetaldehyde (in the presence of NAD+) inhibits the hydrolysis of resorufin acetate only at very high aldehyde concentration. In the absence of cofactor, the rate-limiting step in the hydrolysis of resorufin acetate and of PNP acetate is hydrolysis of the common acetyl-enzyme, as shown by the observation of bursts of chromophoric product and very similar values of kcat. In the presence of NAD+ or NADH, however, the deacylation step with resorufin acetate is greatly accelerated until acylation seems to become rate-limiting, because no burst is seen under these conditions. Millimolar concentrations of Mg2+ activate the hydrolyis of resorufin acetate both in the presence and absence of cofactors. With both Mg2+ and cofactor the kcat for hydrolysis of resorufin acetate is 30–35 s-1; this is three orders of magnitude higher than the kcat for aldehyde oxidation in the presence of Mg2+, showing that the enzyme's potential catalytic efficency is very much hampered by the slowness with which NADH dissociates from its binding site. The pH profile for the hydrolysis of resorufin acetate in the presence of NAD+ or NADH fits well to a theoretical ionization curve of pKa approx. 8.2; it is suggested that this might belong to the enzyme's putative catalytic residue (Cys-302).

BIOSYNTHESE VON STEROIDGLUCURONIDEN BEI VERSCHIEDENEN STOFFWECHSELZUSTÄNDEN

1965 ◽  
Vol 50 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Von Herbert Schriefers ◽  
Brigitte Keck ◽  
Margot Otto
Keyword(s):  
Glycogen Content ◽  
Chemical Nature ◽  
Glucuronic Acid ◽  
Reduction Rate ◽  
Reaction Sequence ◽  
Daily Production ◽  
Rate Limiting Step ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Very High

ABSTRACT A method is described with which the synthesis of steroid glucuronides in the rat liver can be measured directly. Though, as shown in perfusion experiments with testosterone as substrate, the capacity of the liver to synthesize glucuronides is very high (9.5 μg androsterone-glucuronide equivalents/min · g corresponding to a daily production of about 100 mg), its efficiency depends on an undisturbed carbohydrate metabolism: fasting reduces the glucuronide synthesis to one fifth of the control values and alloxan-diabetes causes a decrease of thirty per cent. Intraperitoneal injection of glucose into fasting animals increases the glucuronide synthesis within two hours up to values near the normal. The decline in the rate of glucuronide production cannot be accounted for by the concomitant decrease in testosterone reduction rate. In fasting animals the lowering of the glucuronide synthesis is more pronounced than the ring A reduction rate. On the basis of these facts it is suggested that in the reaction sequence, which proceeds via the formation of dihydro- and tetrahydrocompounds to steroid glucuronides, the transfer of glucuronic acid is the rate limiting step. Obviously the fall in glucuronide synthesis is due to a decrease of liver glucose-6-phosphate and glycogen content, both of which may lead to a diminished supply of UDP-glucuronic acid. Among the aglucones liberated following enzymatic hydrolysis of the chromatographically separated glucuronide fraction, testosterone amounts to only 5 ± 2.04% of the yield expected according to the glucuronic acid estimation. Most of the aglucone fraction consists of products from testosterone hydrogenation. A report dealing with their quantitative distribution and clarifying their chemical nature is in preparation.

scholarly journals Mechanism of d-fructose isomerization by Arthrobacterd-xylose isomerase

1992 ◽  
Vol 283 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Ring Opening ◽  
Ph Dependence ◽  
Xylose Isomerase ◽  
Competitive Inhibitor ◽  
Apparent Dissociation Constant ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Competitive Inhibitors ◽  
Rate Limiting ◽  
Very High

The mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase suggested from X-ray-crystallographic studies was tested by detailed kinetic analysis of the enzyme with various metal ions at different pH values and temperatures. At D-fructose concentrations used in commercial processes Mg2+ is the best activator with an apparent dissociation constant of 63 microM; Co2+ and Mn2+ bind more strongly (apparent Kd 20 microM and 10 microM respectively) but give less activity (45% and 8% respectively). Ca2+ is a strict competitive inhibitor versus Mg2+ (Ki 3 microM) or Co2+ (Ki 105 microM). The kinetics show a compulsory order of binding; Co2+ binds first to Site 2 and then to Site 1; then D-fructose binds at Site 1. At normal concentrations Mg2+ binds at Site 1, then D-fructose and then Mg2+ at Site 2. At very high Mg2+ concentrations (greater than 10 mM) the order is Mg2+ at Site 1, Mg2+ at Site 2, then D-fructose. The turnover rate (kcat.) is controlled by ionization of a residue with apparent pKa at 30 degrees C of 6.0 +/- 0.07 (Mg2+) or 5.3 +/- 0.08 (Co2+) and delta H = 23.5 kJ/mol. This appears to be His-219, which is co-ordinated to M[2]; protonation destroys isomerization by displacing M[2]; Co2+ binds more strongly at Site 2 than Mg2+, so competes more strongly against H+. The inhibition constant (Ki) for the two competitive inhibitors 5-thio-alpha-D-glucopyranose and D-sorbitol is invariant with pH, but Km(app.) in the Mg[1]-enzyme is controlled by ionization of a group with pKa 6.8 +/- 0.07 and delta H = 27 kJ/mol, which appears to be His-53. This shows that Km(app.) is a complex constant that includes the rate of the ring-opening step catalysed by His-53, which explains the pH-dependence. In the Mg[1]Mg[2]-enzyme or Co[1]Co[2]-enzyme, the pKa is lower (6.2 +/- 0.1 or 5.6 +/- 0.08) because of the extra adjacent cation. Hence the results fit the previously proposed pathway, but show that the mechanisms differ for Mg2+ and Co2+ and that the rate-limiting step is isomerization and not ring-opening as previously postulated.

The hydrolysis and biogas production of complex cellulosic substrates using three anaerobic biomass sources

2013 ◽  
Vol 67 (2) ◽  
pp. 293-298 ◽  
Author(s):  
C. Keating ◽  
D. Cysneiros ◽  
T. Mahony ◽  
V. O'Flaherty
Keyword(s):  
Biogas Production ◽  
Positive Role ◽  
Diverse Range ◽  
Factors Affecting ◽  
Rate Limiting Step ◽  
Solid Substrates ◽  
Different Temperatures ◽  
Anaerobic Biomass ◽  
Rate Limiting ◽  
Hydrolysis Of

In this study, the ability of various sludges to digest a diverse range of cellulose and cellulose-derived substrates was assessed at different temperatures to elucidate the factors affecting hydrolysis. For this purpose, the biogas production was monitored and the specific biogas activity (SBA) of the sludges was employed to compare the performance of three anaerobic sludges on the degradation of a variety of complex cellulose sources, across a range of temperatures. The sludge with the highest performance on complex substrates was derived from a full-scale bioreactor treating sewage at 37 °C. Hydrolysis was the rate-limiting step during the degradation of complex substrates. No activity was recorded for the synthetic cellulose compound carboxymethylcellulose (CMC) using any of the sludges tested. Increased temperature led to an increase in hydrolysis rates and thus SBA values. The non-granular nature of the mesophilic sludge played a positive role in the hydrolysis of solid substrates, while the granular sludges proved more effective on the degradation of soluble compounds.

Potential of anaerobic digestion of complex waste(water)

2001 ◽  
Vol 44 (8) ◽  
pp. 115-122 ◽  
Author(s):  
G. Zeeman ◽  
W. Sanders
Keyword(s):  
Waste Water ◽  
Anaerobic Digestion ◽  
Order Relation ◽  
Organic Substrates ◽  
Reactor Technology ◽  
Rate Limiting Step ◽  
Treatment Technologies ◽  
Transport Complex ◽  
Rate Limiting ◽  
Hydrolysis Of

Although they differ greatly in origin complex waste(water)s mainly consist of proteins, lipids, carbohydrates and sometimes lignin in addition. Hydrolysis is the first and generally rate-limiting step in the process of anaerobic digestion of particulate organic substrates. Hydrolysis of particulate polymers can be described by Surface Based Kinetics, but for use in practice the empirical first order relation is advised. Unlike the hydrolysis of protein and carbohydrate, lipid hydrolysis is hardly occurring in the absence of methanogenesis. The latter is probably a physical rather than a biological process and affects the choice for either a one- or a two-step (phase) anaerobic reactor. In the chain of collection and transport, complex wastes often become complex wastewaters simply because of dilution. Dilution not only changes the reactor technology to be applied but also complicates the post-treatment and possibilities for resource recovery. Combining concentrated with diluted waste streams will almost always end up in much more complicated treatment technologies.

Biological treatability of a corn wet mill effluent

2002 ◽  
Vol 45 (12) ◽  
pp. 339-346 ◽  
Author(s):  
G. Eremektar ◽  
O. Karahan-Gul ◽  
F. Germirli-Babuna ◽  
S. Ovez ◽  
H. Uner ◽  
...  
Keyword(s):  
Biological Treatment ◽  
Rate Coefficient ◽  
Activated Sludge Process ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Metabolic Products ◽  
High Level ◽  
Biological Treatability ◽  
Rate Limiting ◽  
Hydrolysis Of

Corn wet mill effluents are studied in terms of their characteristics relevant for biological treatment. They have a high COD of mainly soluble and biodegradable nature, with practically no soluble inert components. They generate a relatively high level of soluble residual metabolic products, which affects the choice of the appropriate biological treatment and favors aerobic activated sludge process. Experimental assessment of process kinetics yields typical values. Hydrolysis of the slowly biodegradable COD, the rate limiting step for the utilization of substrate, is characterized by an overall rate coefficient, which is within the range commonly associated for the hydrolysis of starch.

Growth and cellulolytic activity of Cellulomonas flavigena

1981 ◽  
Vol 27 (12) ◽  
pp. 1260-1266 ◽  
Author(s):  
B. H. Kim ◽  
J. W. T. Wimpenny
Keyword(s):  
Free Enzyme ◽  
Crystalline Cellulose ◽  
Exponential Growth Phase ◽  
Cellulolytic Bacterium ◽  
Cellulolytic Activity ◽  
Mineral Salts ◽  
Cellulomonas Flavigena ◽  
Rate Limiting Step ◽  
Rate Limiting ◽  
Hydrolysis Of

Growth factor requirements, growth kinetics, and the ability to produce the enzyme cellulase were examined in the cellulolytic bacterium Cellulomonas flavigena KIST 321. The organism was found to require only thiamine for growth in mineral salts medium containing simple sugars or cellulose. Growth rates on various carbohydrates suggested that disruption of the crystalline structure was the rate-limiting step in the utilization of crystalline cellulose, and hydrolysis of the polymer itself was as rapid as the uptake of the hydrolytic product. When the organism was grown on cellulose the cellulolytic activity appeared to be bound to the cell at the beginning of the exponential growth phase: only after this did cell-free enzyme activity appear. The cell-free enzyme appeared to be unstable, and its activity decreased at the beginning of the stationary phase.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

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