The Enzymatically Catalyzed Mutarotation. The Mechanism of Action of Mutarotase (Aldose 1-Epimerase) from Escherichia coli

Galactose mutarotase plays a key role in normal galactose metabolism by catalyzing the interconversion of ␤-D-galactose and ␣-D-galactose. Here we describe the three-dimensional architecture of galactose mutarotase from Lactococcus lactis determined to 1.9-Å resolution. Each subunit of the dimeric enzyme displays a distinctive ␤-sandwich motif. This tertiary structural element was first identified in ␤-galactosidase and subsequently observed in copper amine oxidase, hyaluronate lyase, chondroitinase, and maltose phosphorylase.

mentioning

confidence: 99%

mentioning

confidence: 99%

High Resolution X-ray Structure of Galactose Mutarotase from Lactococcus lactis

Thoden

Holden

2002

“…A possible catalytic mechanism for galactose mutarotase, first suggested by Hucho and Wallenfels (15) and outlined in Scheme 1, involves the abstraction of a proton from the C-1 hydroxyl group of the sugar by an enzymatic base and donation of a proton to the C-5 oxygen by a side chain acidic group thus resulting in ring cleavage. Subsequent rotation about the C-1/C-2 bond, followed by abstraction of the proton on the C-5 oxygen and donation of a proton back to the C-1 oxygen leads to product formation.…”

mentioning

confidence: 99%

Structural and Kinetic Studies of Sugar Binding to Galactose Mutarotase from Lactococcus lactis

Thoden¹,

Kim²,

Raushel³

et al. 2002

Galactose mutarotase catalyzes the conversion of ␤-Dgalactose to ␣-D-galactose in the Leloir pathway for galactose metabolism. The high resolution x-ray structure of the dimeric enzyme from Lactococcus lactis was recently solved and shown to be topologically similar to the 18-stranded, anti-parallel ␤-motif observed for domain 5 of ␤-galactosidase. In addition to determining the overall molecular fold of galactose mutarotase, this initial investigation also provided a detailed description of the electrostatic interactions between the enzyme and its physiologically relevant substrate, galactose. Specifically, the side chains of His-96 and His-170 were shown to be located within hydrogen bonding distance to the C-5 oxygen of the substrate, while the carboxylate of

“…Five highly conserved residues (Arg-71, His-96, His-170, Asp-243, and Glu-304) were within hydrogen-bonding distance to the hydroxyl groups of the bound galactose ligand. Previous kinetic studies suggested that the reaction mechanism for the enzyme proceeds via an acid/base mechanism with a transient ring opening (26,27), and the model of the mutarotase from L. lactis implicated Glu-304 as the likely catalytic base and either His-96 or His-170 as the catalytic acid (24). Accordingly, the reaction mechanism of galactose mutarotase is thought to occur via abstraction of the proton from the C-1 hydroxyl group by Glu-304 in the L. lactis enzyme and protonation of the ring oxygen of the sugar by His-170, which results in ring opening.…”

mentioning

confidence: 99%

Molecular Structure of Human Galactose Mutarotase

Thoden

Timson

Reece

et al. 2004

Galactose mutarotase catalyzes the conversion of ␤-Dgalactose to ␣-D-galactose during normal galactose metabolism. The enzyme has been isolated from bacteria, plants, and animals and is present in the cytoplasm of most cells. Here we report the x-ray crystallographic analysis of human galactose mutarotase both in the apoform and complexed with its substrate, ␤-D-galactose. The polypeptide chain folds into an intricate array of 29 ␤-strands, 25 classical reverse turns, and 2 small ␣-helices. There are two cis-peptide bonds at Arg-78 and Pro-103. The sugar ligand sits in a shallow cleft and is surrounded by Asn-81, Arg-82, His-107, His-176, Asp-243, Gln-279, and Glu-307. Both the side chains of Glu-307 and His-176 are in the proper location to act as a catalytic base and a catalytic acid, respectively. These residues are absolutely conserved among galactose mutarotases. To date, x-ray models for three mutarotases have now been reported, namely that described here and those from Lactococcus lactis and Caenorhabditis elegans. The molecular architectures of these enzymes differ primarily in the loop regions connecting the first two ␤-strands. In the human protein, there are six extra residues in the loop compared with the bacterial protein for an approximate longer length of 9 Å. In the C. elegans protein, the first 17 residues are missing, thereby reducing the total number of ␤-strands by one.During normal galactose metabolism, ␤-D-galactose is converted to glucose 1-phosphate via the action of four enzymes that constitute the Leloir pathway as shown in Scheme 1 (1). In the first step of this pathway, ␤-D-galactose is epimerized to ␣-D-galactose through the action of galactose mutarotase. The second step involves the phosphorylation of ␣-D-galactose to galactose 1-phosphate by galactokinase. As indicated in Scheme 1, galactose 1-phosphate uridylyltransferase catalyzes the third step by transferring a UMP group from UDP-glucose to galactose 1-phosphate, thereby generating glucose 1-phosphate and UDP-galactose. To complete the pathway, UDPgalactose is converted to UDP-glucose by UDP-galactose 4-epimerase.Mutations in three of the enzymes of the Leloir pathway, namely galactokinase, galactose 1-phosphate uridylyltransferase, or UDP-galactose 4-epimerase, have been demonstrated to result in the diseased state known as galactosemia (2, 3). The symptoms of this genetic disease include early onset cataracts (typically within the first two years of life) and, in more severe cases, liver, kidney, and brain damage. Cataract formation is believed to be caused by the buildup of unmetabolized galactose in the lens of the eye. Aldose reductase catalyzes the reduction of the sugar to galactitol (dulcitol), which is not, in contrast to galactose, readily transported across the plasma membrane (4 -6). As such, the accumulation of this highly osmotically active compound draws excess water into the lens resulting in cataracts by a mechanism that is not fully understood. The more severe symptoms of galactosemia are most likely caused b...