21 Biological
Mechanisms
Formation of Hydrogen
Deoxy
Involved
in
the
Sugars: E n z y m a t i c
Mediation
Downloaded by RUTGERS UNIV on December 28, 2017 | http://pubs.acs.org Publication Date: June 1, 1973 | doi: 10.1021/ba-1971-0117.ch021
A Possible Example for the Evolutionary Process of Enzyme Catalysis OTHMAR GABRIEL
a
Department of Biochemistry, Georgetown University Schools of Medicine and Dentistry, Washington, D.C. 20007 For the purpose of orientation, an overall view of deoxyhexose biosynthesis is provided with examples of some of the well documented enzymatic reactions. Detailed studies of the reaction mechanism of deoxy sugar biosynthesis are reported with special emphasis upon enzymatic hydrogen transfer reactions, substrate conformation, and coenzyme participation. Extension of these studies to enzymes involved in various other sugar transformations reveal close similarities in the mechanism of hydrogen mediation for several enzymes. As a result, some unifying principles for enzyme catalyzed hydrogen mediation are established. The objective of this approach is to provide eventually a rational basis for an understanding of the close relation existing between substrate conformation and stereochemical changes of protein conformation at the active site during enzyme catalyzed transformations. T^Veoxy sugars can be defined as sugars with one or more alcoholic hydroxy groups in a monosaccharide replaced by hydrogen(s). For example, the naturally-occurring pentose, D-ribose, is also found as its correponding deoxypentose, 2-deoxy-D-ribose. W e are aware of the biological significance caused by this substitution and the fundamental difference in the metabolism and role of ribonucleic acid and deoxyPresent address: Boston, Mass. 02114. a
Harvard Medical School, Massachusetts General Hospital,
387 Isbell; Carbohydrates in Solution Advances in Chemistry; American Chemical Society: Washington, DC, 1973.
Downloaded by RUTGERS UNIV on December 28, 2017 | http://pubs.acs.org Publication Date: June 1, 1973 | doi: 10.1021/ba-1971-0117.ch021
388
CARBOHYDRATES IN SOLUTION
ribonucleic acid, respectively. It is, therefore, not surprising to find that a similar disparity of metabolic fate and function also exists between hexoses and deoxyhexoses. W h i l e the major function of naturally-occurring hexoses is to provide the necessary energy for the living processes of the cell, deoxyhexoses are not involved in this process. Deoxy sugars have been recognized as immunological determinants when attached to macromolecules and are responsible for the specificity of immune reactions. The attachment of various deoxyhexoses to strategic points on macromolecules imply far reaching biological consequences for our understanding of problems ranging from our natural defense mechanisms to cell surface and membrane interactions. Consequently, attention has been paid to the elucidation of pathways leading to the formation of these important sugar derivatives. For the purpose of orientation, the metabolic pathways of some of the naturally-occurring deoxy sugars is presented. This is followed by a detailed study of the enzyme reaction mechanism with emphasis on hydrogen mediation and its relation to substrate conformation and stability. The picture emerging from these experiments is compared with studies carried out by other investigators on several different enzymatic systems not related to deoxyhexose biosynthesis. W e hope to show similarities in the mechanism of hydrogen mediation common to several enzymatic reactions, thereby indicating a more general applicability of the reported findings. Metabolic Pathways 6-Deoxyhexoses. The interconversions leading to the biosynthesis of 6-deoxyhexoses occur at the level of sugar nucleotides. The first conversion of this type, GDP-D-mannose to GDP-L-fucose (GDP-6-deoxy-Lgalactose), was reported by Ginsburg (1,2). Similarly, TDP-D-glucose is transformed into TDP-L-rhamnose (3, 4, 5) (TDP-6-deoxy-L-mannose). The strategy for the biosynthesis of 6-deoxyhexoses is indicated i n Figure 1. In each instance the first stage is the formation of a nucleotide-linked 4-keto-6-deoxy intermediate. The synthesis of the 4-keto derivative is common to all systems of biosynthesis of deoxyhexoses described thus far. The reaction is catalyzed by enzymes referred to as oxidoreductases and is irreversible. Once a hexose becomes converted to the nucleotide4-keto-derivative, it is no longer available for use by the main metabolic pathways or for energy production. It is converted to a nucleotide linked deoxyhexose, which serves as a precursor for incorporation into such macromolecules as glycoproteins. It is advantageous for the following discussion to emphasize the key role of the 4-keto-intermediate as common to all deoxyhexose biosynthetic pathways. The formation of a 4-
Isbell; Carbohydrates in Solution Advances in Chemistry; American Chemical Society: Washington, DC, 1973.
21.
GABRIEL
Deoxy Sugar Biosynthesis
389
Downloaded by RUTGERS UNIV on December 28, 2017 | http://pubs.acs.org Publication Date: June 1, 1973 | doi: 10.1021/ba-1971-0117.ch021
ulose is an organic synthesis step chosen by nature to accomplish the necessary transformations. Epimerizations at carbons 3 and 5 of the hexose moiety followed by stereospecific reduction at carbon-4 can lead to a variety of deoxy sugars. M a n y of these biosynthetic systems have been elucidated, and some of the established pathways are listed in Table I. A l l take advantage of a similar principle and follow the scheme outlined above. Also, the same sugar may be conjugated with different purine or pyrimidine bases.
0-MiMOse
Figure 1.
Biosynthesis of 6-deoxyhexoses
3,6-Dideoxyhexoses. Several bacterial antigenic determinants with the general structure of 3,6-dideoxyhexoses occur in the cell wall of Pasteurella and Salmonella strains. Most of the transformations reported so far occur as cytidine nucleotides (see Table I, References 15, 16, 17, 18, 19). Here, again the first step is the transformation of the cytidine diphospho-linked glucose into its corresponding 4-keto derivative. B y at least two distinct steps, requiring N A D P H , reduction to several different 3,6-dideoxyhexoses have been reported. One 3,6-dideoxyhexose C D P tyvelose (3,6-dideoxy-D-arabino hexose) is formed by a specific 2-epimerase from CDP-paratose (24). Acetamidodeoxyhexoses. A further modification of the 4-keto-intermediate has been independently shown by Ashwell and by Strominger and associates (Table I, References 20, 21, 22, 23). Transamination reactions with L-glutamate as the amino donor and pyridoxal phosphate as coenzyme led to formation of 3-amino 3,6-dideoxy- and 4-amino 4,6dideoxyhexoses, respectively. Acetylation with acetyl coenzyme A yields the naturally-occurring N-acetyl amino sugar derivatives.
Isbell; Carbohydrates in Solution Advances in Chemistry; American Chemical Society: Washington, DC, 1973.
390
CARBOHYDRATES IN SOLUTION
Downloaded by RUTGERS UNIV on December 28, 2017 | http://pubs.acs.org Publication Date: June 1, 1973 | doi: 10.1021/ba-1971-0117.ch021
Detailed Reaction Mechanism of Deoxy Sugar Biosynthesis TDPG-oxido Reductase. The enzyme initiating deoxyhexose biosynthesis i n E. coli, TDPG-oxidoreductase, was selected as a model for a careful study of its reaction mechanism. As mentioned before, the molecular rearrangement leading to the formation of a 4-keto-intermediate is common to a l l 6-deoxyhexose pathways. It involves oxidation at carbon-4 and conversion of the primary alcoholic group at carbon-6 to a methyl group. The elucidation of the reaction mechanism was accomplished by using three different approaches: 1) a study of properties of model compounds to mimic the action of the enzyme, 2) preparation of selectively tritiated substrates to trace the fate of the tritium during the enzymatic reaction, and 3) isolation of the enzyme protein as a homogeneous component and studies with the pure protein.
Table I. Oxidoreductase Sugar Nucleotide Nucleoside Diphosphohexose
Enzyme Systems for
4-Keto Intermediate 6-Deoxyhexoses
Guanosine
D-Mannose
Uridine
D-Glucose
Thymidine
D-Glucose
Guanosine
D-Mannose
Guanosine
D-Mannose
Thymidine
D-Glucose
Guanosine
D-Mannose
Cytidine
D-Glucose
Cytidine
D-Glucose
Cytidine
D-Glucose
Thymidine
D-Glucose
Thymidine
D-Glucose
GDP-6-deoxy-D-lyxo-4-hexulose (4-keto-6-deoxy-D-mannose) UDP-6-deoxy-D-xylo-4-hexulose (4-keto-6-deoxy-D-glucose) TDP-6-deoxy-D-xylo-4-hexulose (4-keto-6-deoxy-D-glucose) GDP-6-deoxy-D-lyxo-4-hexulose (4-keto-6-deoxy-mannose) GDP-6-deoxy-D-lyxo-4-hexulose (4-keto-6-deoxy-mannose) TDP-6-deoxy-D-xylo-4-hexulose (4-keto-6-deoxy-D-glucose)
3, 6-Dideoxyhexoses GDP-6-deoxy-D-xylo-4-hexulose (4-keto-6-deoxy-glucose) CDP-6-deoxy-D-xylo-4-hexulose (4-keto-6-deoxy-glucose) CDP-6-deoxy-D-xylo-4-hexulose (4-keto-6-deoxy-glucose) CDP-6-deoxy-D-xylo-4-hexulose (4-keto-6-deoxy-glucose)
6-Deoxy Amino Sugars TDP-6-deoxy-D-xylo-4-hexulose (4-keto-6-deoxy-glucose) TDP-6-deoxy-D-xylo-4-hexulose (4-keto-6-deoxy-glucose)
Isbell; Carbohydrates in Solution Advances in Chemistry; American Chemical Society: Washington, DC, 1973.
21.
GABRIEL
391
Deoxy Sugar Biosynthesis
The major phases of our experiments are presented below. To facilitate the discussion, a summary of our findings is shown i n Figure 2, indicating the mechanism involved. TDP-D-glucose is initially attacked by e n z y m e - N A D (enzyme protein containing one mole of firmly bound N A D ) at carbon 4 to yield TDP-D-xylo-4-hexulose and accompanying formation of enzyme N A D H . The 4-ulose rearranges by /^-elimination of water between carbons 5 and 6 to form an unsaturated glucoseen. This unsaturated 5,6-glucoseen serves as hydrogen acceptor for e n z y m e - N A D H to restore e n z y m e - N A D and leads to the end product of the reaction TDP-6-deoxy-D-xylo-4hexulose. +
+
Downloaded by RUTGERS UNIV on December 28, 2017 | http://pubs.acs.org Publication Date: June 1, 1973 | doi: 10.1021/ba-1971-0117.ch021
+
Returning to the first phase of our studies on the detailed reaction mechanism, we discuss some model experiments which were carried out
6-Deoxyhexose Biosynthesis Epimerizations of 4-Keto Intermediate C3 and C5 C3 and C5 C3 and C5 None None C3 and C5
C3? and C 5 None None C3? and C5
None None
NADPH > E n d Product
GDP-6-deoxy-L-galactose (L-Fucose) (1, 6) UDP-6-deoxy-L-mannose (L-Rhamnose) (7) TDP-6-deoxy-L-mannose (L-Rhamnose) (3, 4, 5, 8) GDP-6-deoxy-D-mannose (D-Rhamnose) (9, 10, 11, 12, 13) GDP-6-deoxy-D-talose (9, 12, 13) TDP-6-deoxy-L-talose (H) G D P - 3 , 6-dideoxy-L-xylohexose (Colitose) 15, 16) C D P - 3 , 6-dideoxy-D-ribohexose (Paratose) (17, 18, 19) C D P - 3 , 6-dideoxy-D-xylohexose (Abequose) (17, 18, 19) C D P - 3 , 6-dideoxy-L-arabinohexose (Ascarylose) (17) TDP-3-acetamino-3, 6-dideoxy-D-galactose (20) TDP-4-acetamido-4, 6-dideoxy-D-glucose (21, 22, 23)
Isbell; Carbohydrates in Solution Advances in Chemistry; American Chemical Society: Washington, DC, 1973.
392
CARBOHYDRATES IN SOLUTION
Downloaded by RUTGERS UNIV on December 28, 2017 | http://pubs.acs.org Publication Date: June 1, 1973 | doi: 10.1021/ba-1971-0117.ch021
to get insight into the stability of the 4-ulose derivative, a postulated intermediate of the enzymatic reaction.
bigure 2.
Reaction mechanism for TDPG oxidoreductase
It is well established that oxygen i n the presence of platinum (Adams catalyst) can achieve specific oxidation of secondary alcohols by a preferential attack upon hydrogen in an equatorial position (25). Catalytic oxidation of methyl
