86
P. K. STUMPF AND G. A. BARBER
Both 3-ethylenic pentenoyl and 3-ethylenic hexenoyl thioesters are inert.
The reaction appears to be irreversible, since crotonyl thioesters do
not convert to vinylacetyl thioesters. The equilibrium for the alkalicatalyzed tautomerization of vinylacetic acid to crotonate is greater
than 99% in favor of crotonate. This enzyme, first reported in C.
kluyveri, is also found in Clostridium tetanomorphum and in sheep
liver homogenate (31). If the crystalline crotonase prepared by Stern
were contaminated with small amounts of this isomerase, it could explain the low order of activity of crotonase for the 3-ethylenic acyl
thioesters which Stern has observed. Crystallinity of an enzyme protein
does not guarantee purity.
c. Fumarase. Just as acyl dehydrogenases have as their counterpart
succinic dehydrogenase so crotonase has as its counterpart fumarase.
Like crotonase, no cofactors have been found associated with the
crystalline enzyme. Only two substrates are active, c/s-fumaric acid
and L-malic acid. The enzyme is widespread in different phyla. It has
been prepared in the crystalline form and studied extensively.
When the hydration of fumaric acid to L-malic acid is catalyzed
in D 2 0 by fumarase, L-malate contains only one deuterium atom per
molecule which is not readily exchangeable into the medium (40, 41).
Since fumarate does not incorporate deuterium, the addition of hydrogen is stereospecific as is the OH incorporation. When monodeuterated malate is dehydrated enzymatically in water, the V max and
K m is the same as for ordinary L-malate. If the breaking of the methylene C—H bond were rate determining, substitution of deuterium for H
would produce a decrease in rate. Absence of a deuterium rate effect
in the dehydration of monodeutero-L-malate indicates that the exchange of a specific H atom with the H atom of the medium proceeds
at a much faster rate than the OH participation. Thus Alberty pictures
the reaction as shown (Eq. 18).
Enz + Malic ^± Enz-malic ^=± Enz-X ^=± Enz-fumaric ^ Enz + Fumaric (18)
P. K. STUMPF AND G. A. BARBER
Both 3-ethylenic pentenoyl and 3-ethylenic hexenoyl thioesters are inert.
The reaction appears to be irreversible, since crotonyl thioesters do
not convert to vinylacetyl thioesters. The equilibrium for the alkalicatalyzed tautomerization of vinylacetic acid to crotonate is greater
than 99% in favor of crotonate. This enzyme, first reported in C.
kluyveri, is also found in Clostridium tetanomorphum and in sheep
liver homogenate (31). If the crystalline crotonase prepared by Stern
were contaminated with small amounts of this isomerase, it could explain the low order of activity of crotonase for the 3-ethylenic acyl
thioesters which Stern has observed. Crystallinity of an enzyme protein
does not guarantee purity.
c. Fumarase. Just as acyl dehydrogenases have as their counterpart
succinic dehydrogenase so crotonase has as its counterpart fumarase.
Like crotonase, no cofactors have been found associated with the
crystalline enzyme. Only two substrates are active, c/s-fumaric acid
and L-malic acid. The enzyme is widespread in different phyla. It has
been prepared in the crystalline form and studied extensively.
When the hydration of fumaric acid to L-malic acid is catalyzed
in D 2 0 by fumarase, L-malate contains only one deuterium atom per
molecule which is not readily exchangeable into the medium (40, 41).
Since fumarate does not incorporate deuterium, the addition of hydrogen is stereospecific as is the OH incorporation. When monodeuterated malate is dehydrated enzymatically in water, the V max and
K m is the same as for ordinary L-malate. If the breaking of the methylene C—H bond were rate determining, substitution of deuterium for H
would produce a decrease in rate. Absence of a deuterium rate effect
in the dehydration of monodeutero-L-malate indicates that the exchange of a specific H atom with the H atom of the medium proceeds
at a much faster rate than the OH participation. Thus Alberty pictures
the reaction as shown (Eq. 18).
Enz + Malic ^± Enz-malic ^=± Enz-X ^=± Enz-fumaric ^ Enz + Fumaric (18)