Aldolase

I.U.B.: 4.1.2.13
D-Fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase

Fructose-biphosphate aldolase (ALD) catalyzes a key reaction in glycolysis and energy production:

Aldolase is present in all animal and plant tissue and in most microorganisms. Class I aldolase, found in animal and higher plant tissue, is characterized by not requiring a bivalent metal cofactor and by the formation of a ketimine Schiff base intermediate with the substrate dihydroxyacetone phosphate. (This base may be reduced by NaBH₄ and the enzyme inactivated). Class II aldolase, found in primitive cells such as yeast and bacteria, requires a metal cofactor and is inhibited by EDTA. See Heron and Caprioli (1975), London (1974) and Lebherz et al. (1973).

In animal tissue there are three types of aldolases: Type A, the major form, is found in muscle; Type B in liver and kidney; and Type C (plus some A) in brain. The enzyme is tetrameric and may exist as five different isozymes (for example: A4, A3B, A2B2, AB3 and B4) which may, to various degrees, be organ specific. See Lebherz and Rutter (1969) and Penhoet et al. (1967), also Lee et al. (1975).

The aldolases have been reviewed in detail by Horecker et al. (1972).

The enzyme is used for the preparation of dihydroxyacetone phosphate, D-glyceraldehyde-3-phosphate, and condensation products from dihydroxyacetone phosphate and aldehydes. It is also used to determine the concentration of D-fructose-1,6-bisphosphate.

Characteristics of Aldolase from Rabbit Muscle :

Molecular weight: 161,000 ± 3,000.

Subunits: approx. 40,000 (Szuchet and Yphanis 1973; Sia and Horecker 1968).

Composition: The molecule is composed of four subunits (Penhoet et al. 1967). Each subunit is reported to make an independent contribution to the activity (Brenner-Holzack and Leuthardt 1972). The amino acid composition has been given by Anderson et al. (1969). Eagles et al. (1969) have reported on subunit structure. See also: Heidner et al. (1971); Chan and Mawer (1972); Hsu and Neet (1973); Salter et al. (1973); Szuchet and Yphantis (1973); Lai et al. (1974); Vimard et al. (1975). Active site conformations have been reported on by Crowder et al. (1973), Grazi et al. (1973), Hartman et al. (1973), and Anderson and Kaplan (1974).

Optimum pH: 7.0 (Taylor 1955).

Extinction coefficient: = 9.38 (Donovan 1964).

Isoelectric Point: pH 6.1 (0.1 M phosphate buffer) ( Taylor 1955).

Specificity: The enzyme is markedly specific for aldol condensation (Rutter 1961). In the forward reaction it reacts with β-D-fructofuranose-1,6-bisphosphate and fructose-1-phosphate (Wurster and Hess 1973 and Rutter et al. 1961).

Inhibitors: Cu²⁺, Ag⁺ and Zn²⁺ ions, o-phenanthroline (Kobashi and Horecker 1967). ATP>ADP>AMP (Spolter et al. 1965). It is inactivated by N-bromoacetylethanolamine phosphate (Hartman et al. 1973) and pyridoxal phosphate (Davis et al. 1971).

Stability: The enzyme is irreversibly denatured at pH values less than 4.5 (Rutter 1961). A crystalline suspension in ammonium sulfate solution, pH 7.6, is stable for at least six months at 4°C.