CHAPTER 6 . Organic Chemical Reaction Rates in the Ocean:
145
LYA-Iysine
LYA-£-amino-a-ketocaproic acid
LYA-S-aminovaleric acid
Fig. 6.4. Oxidative deamination of LYA-lysine
1. Fluorescent analogs can be detected by High Pressure Liquid Chromatography
(HPLC) with an on-line fluorometer. Therefore, the disappearance of the added substrate from a seawater sample and concurrent production of the oxidation product
can be followed with time, allowing the estimation of oxidation rates.
2. The size of the probes precludes incorporation across the cell membrane of microorganisms, therefore only extracellular reactions are detected.
3. The detection limit is about 5 nM, which is in the range of (or lower than) the concentration of an individual amino acid in sea water.
4. They are stable to chemical reactions on the time scale of the biological reactions
we are measuring (days to weeks).
5. Experiments determined that natural L-amino acids compete with the fluorescent
derivative suggesting similarity between substrates as explained below.
In a phytoplankton culture, competitive inhibition of the oxidation of LYA-lysine
was observed in the presence of the mixture of L-alanine and L-isoleucine (0.5 flM
each) (Fig. 6.5). The half-saturation constant (Ks) of the oxidation of LYA-lysine increased upon the addition of these amino acids from 0.11 flM (uninhibited) to 0.76 flM
(inhibited). The maximum velocity (Vm) of the oxidation remains unaltered (about
0.6 nM min -1) in both cases, as the regression lines share the same y-intercept (1 / V m).
Competitive inhibition is demonstrated as an increase in Ks of an enzymatic reaction
in the presence of an inhibitor (Dixon and Webb 1958). Ks increases because a larger
amount of substrate is needed to convert the enzyme to the enzyme-substrate mixture in the presence of the inhibitor. On the other hand, since the influence of the competitive inhibitor is less at high substrate concentration, V m is independent of the presence of competitors (Dixon and Webb 1958). In summary, L-amino acid oxidases apparently do not discriminate between natural amino acids and LYA-lysine.
145
LYA-Iysine
LYA-£-amino-a-ketocaproic acid
LYA-S-aminovaleric acid
Fig. 6.4. Oxidative deamination of LYA-lysine
1. Fluorescent analogs can be detected by High Pressure Liquid Chromatography
(HPLC) with an on-line fluorometer. Therefore, the disappearance of the added substrate from a seawater sample and concurrent production of the oxidation product
can be followed with time, allowing the estimation of oxidation rates.
2. The size of the probes precludes incorporation across the cell membrane of microorganisms, therefore only extracellular reactions are detected.
3. The detection limit is about 5 nM, which is in the range of (or lower than) the concentration of an individual amino acid in sea water.
4. They are stable to chemical reactions on the time scale of the biological reactions
we are measuring (days to weeks).
5. Experiments determined that natural L-amino acids compete with the fluorescent
derivative suggesting similarity between substrates as explained below.
In a phytoplankton culture, competitive inhibition of the oxidation of LYA-lysine
was observed in the presence of the mixture of L-alanine and L-isoleucine (0.5 flM
each) (Fig. 6.5). The half-saturation constant (Ks) of the oxidation of LYA-lysine increased upon the addition of these amino acids from 0.11 flM (uninhibited) to 0.76 flM
(inhibited). The maximum velocity (Vm) of the oxidation remains unaltered (about
0.6 nM min -1) in both cases, as the regression lines share the same y-intercept (1 / V m).
Competitive inhibition is demonstrated as an increase in Ks of an enzymatic reaction
in the presence of an inhibitor (Dixon and Webb 1958). Ks increases because a larger
amount of substrate is needed to convert the enzyme to the enzyme-substrate mixture in the presence of the inhibitor. On the other hand, since the influence of the competitive inhibitor is less at high substrate concentration, V m is independent of the presence of competitors (Dixon and Webb 1958). In summary, L-amino acid oxidases apparently do not discriminate between natural amino acids and LYA-lysine.