Inhibition by oxalates of spinach chloroplast phenolase in unfrozen and frozen states

Spinach chloroplast phenolase was inhibited by oxalic acid and its salts. Complete inhibitions were induced instantly in the acidic region (e.g. by 1 and 5 mM oxalate at pH 5 and 5.5, respectively), and in the neutral region pre-incubation of the enzyme with oxalates could also lead to complete loss of activity. The inhibition mode was non-competitive for phenol substrate with K_i of 0.9 mM pH 6.8. Reduction of enzyme activity in a crude extract of chloroplasts induced by freezing at neutral pH was due to the presence of ammonium oxalate. With 0.5 mM oxalate, the inhibition attained 75% under frozen conditions, whilst no inhibition could be detected in the enzyme which had not been frozen. Free oxalic acid and K⁺ and Na⁺ salts also caused freezing inhibition. Glyoxylic and oxamic acids acted as inhibitors with less efficiency. With a pure mushroom tyrosinase (phenolase), essentially the identical results were obtained using the same conditions.