scholarly journals Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5′-phosphate. Demonstration of an essential lysine residue

1984 ◽  
Vol 222 (1) ◽  
pp. 93-102 ◽  
Author(s):  
G J Hart ◽  
F J Leeper ◽  
A R Battersby
Keyword(s):  
Euglena Gracilis ◽  
Enzymic Activity ◽  
Lysine Residue ◽  
Porphobilinogen Deaminase ◽  
Amino Group ◽  
Spectroscopic Evidence ◽  
Lysine Residues ◽  
Modified Enzyme ◽  
Rhodopseudomonas Spheroides ◽  
Covalently Bound

When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5′-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5′-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5′-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.

scholarly journals Horse liver alcohol dehydrogenase. A study of the essential lysine residue

1975 ◽  
Vol 149 (3) ◽  
pp. 627-635 ◽  
Author(s):  
S S Chen ◽  
P C Engel
Keyword(s):  
Alcohol Dehydrogenase ◽  
Rate Constant ◽  
Lysine Residue ◽  
Horse Liver Alcohol Dehydrogenase ◽  
First Order ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver ◽  
Lysine Residues ◽  
Modified Enzyme ◽  
Covalent Complex

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10°C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.

scholarly journals Mechanism of action of porphobilinogen deaminase. The participation of stable enzyme substrate covalent intermediates between porphobilinogen and the porphobilinogen deaminase from Rhodopseudomonas spheroides

1981 ◽  
Vol 195 (1) ◽  
pp. 177-181 ◽  
Author(s):  
P M Jordan ◽  
A Berry
Keyword(s):  
Mechanism Of Action ◽  
Catalytic Cycle ◽  
Porphobilinogen Deaminase ◽  
Enzyme Substrate ◽  
Enzyme Intermediates ◽  
Rhodopseudomonas Spheroides ◽  
Covalently Bound

Highly stable labelled complexes are formed between porphobilinogen deaminase and stoicheiometric amounts of [14C]porphobilinogen. On completion of the catalytic cycle by the addition of excess of substrate, the complexes yield labelled product and display all the properties expected from covalently bound enzyme intermediates involved in the deaminase catalytic sequence.

The Reaction of Aminoacylase with Chloromethylketone Analogs of Amino Acids

1977 ◽  
Vol 32 (9-10) ◽  
pp. 769-776 ◽  
Author(s):  
Jürgen Frey ◽  
Werner Kordel ◽  
Friedhelm Schneider
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Active Site ◽  
Lysine Residue ◽  
Amino Group ◽  
Sh Groups ◽  
Competitive Inhibitors ◽  
Kinetics Of ◽  
Covalently Bound

Abstract1. Aminoacylase is irreversibly inactivated by the chloromethylketone analogs of benzyloxy-carbonyl-L-alanine, L-alanine, L-leucine, L-aspartic acid (β), tosyl-L-phenylalanine and L-leucyl-L-alanine. The kinetics of the inactivation of the enzyme by the halo-methylketones were investigated.2. Leucyl-and alanyl chloromethylketone inactivate the enzyme by blocking of 4 SH groups. Experiments with [U-14C] leucyl chloromethylketone confirm that maximal 4 residues are covalently bound to the protein.3. Inactivation of the enzyme by benzyloxycarbonylalanyl and tosylphenylalanyl chloromethyl­ ketone is the result of the substitution of the £-amino group of one lysine residue per active site and not of SH groups. However, in the presence of competitive inhibitors these halomethylketones react only with the SH groups of the enzyme, too.

The Action of Thrombin on Modified Fibrinogen

1983 ◽  
Vol 49 (03) ◽  
pp. 208-213
Author(s):  
A J Osbahr
Keyword(s):  
Physical Properties ◽  
Negative Charge ◽  
Lysine Residue ◽  
Fibrinopeptide A ◽  
Amino Groups ◽  
Lysine Residues ◽  
Citraconic Anhydride

SummaryThe modification of canine fibrinogen with citraconic anhydride modified the ε-amino groups of the fibrinogen and at the same time generated additional negative charges into the protein. The addition of thrombin to the modified fibrinogen did not induce polymerization; however, the fibrinopeptide was released at a faster rate than from the unmodified fibrinogen. The physical properties of the citraconylated fibrinogen were markedly altered by the modification of 50-60 lysine residues in one hour. A modified fibrinopeptide-A was released by thrombin from the modified fibrinogen and was electrophoretically more anionic than the unmodified fibrinopeptide-A. Edman analysis confirmed the modification of the lysine residue present in the peptide. The rate of removal of citraconylated fibrinopeptide-A from modified fibrinogen by thrombin was 30 to 40 percent greater than the cleavage of unmodified fibrinopeptide-A from unmodified fibrinogen. However, the modification of 60 or more lysine residues in the fibrinogen produced a decrease in the rate of cleavage of citraconylated fibrinopeptide-A. The results suggest that additional negative charge in the vicinity of the attachment of fibrinopeptide-A to canine fibrinogen aids in the removal of the peptide by thrombin.

Comparison of Different Methods for the Determination of Phenylalanine Hydroxylase Activity in Rat Liver and Euglena gracilis

1984 ◽  
Vol 39 (7-8) ◽  
pp. 728-733 ◽  
Author(s):  
Rita M. Fink ◽  
Erich F. Elstner
Keyword(s):  
Rat Liver ◽  
Euglena Gracilis ◽  
Phenylalanine Hydroxylase ◽  
Hplc Method ◽  
Enzymic Activity ◽  
Hydroxylase Activity ◽  
Product Separation ◽  
Tyrosine Concentration ◽  
Layer Chromatography

Abstract Three different methods for the determination of phenylalanine hydroxylase activity have been compared: a) Differential photometric assay of the increase in tyrosine concentration in the presence of phenylalanine; b) Product separation by thin layer chromatography and scintillation counting of the [14C]tyrosine formed;c) HPLC separation and spectrofluorometric quantification of derivatized amino acids. A comparison of the activities of phenylalanine hydroxylase in rat liver and Euglena gracilis clearly showed that only rat liver contains this enzymic activity as shown by methods b) and c) although pseudo-activity of Euglena gracilis preparations was found during the spectrophotometric test a). The HPLC method proved to be the fastest, most reliable and convenient method for direct tyrosine determination and thus for measuring phenylalanine hydroxylase activity.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

2018 ◽  
Vol 118 (02) ◽  
pp. 340-350 ◽  
Author(s):  
Ingrid Stroo ◽  
J. Marquart ◽  
Kamran Bakhtiari ◽  
Tom Plug ◽  
Alexander Meijer ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Active Form ◽  
Coagulation Factor ◽  
Lysine Residue ◽  
Factor Ix ◽  
Factor Xi ◽  
Lysine Residues ◽  
Factor Xia

AbstractCoagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

Amino-terminal groups in zymogen activation: effect of nitrous acid on pepsin

1969 ◽  
Vol 47 (12) ◽  
pp. 1099-1101 ◽  
Author(s):  
T. Hofmann
Keyword(s):  
Nitrous Acid ◽  
Lysine Residue ◽  
Amino Group ◽  
Serine Proteinases ◽  
Zymogen Activation ◽  
Activation Effect ◽  
Amino Terminal ◽  
Tryptophan Residues ◽  
Terminal Amino ◽  
Isoleucine Residue

Nitrous acid rapidly deaminates the N-terminal isoleucine residue of pepsin. The enzyme retains more than 60% of its activity. The loss of 30–40% activity can be ascribed to the reaction of two tryptophan residues with nitrous acid. The single lysine residue is also deaminated. These results are in contrast to those obtained with serine proteinases where nitrous acid causes inactivation due to the deamination of the N-terminal amino group.

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