MECHANISM OF THE REACTION CATALYZED BY MANDELATE RACEMASE. 2. CRYSTAL STRUCTURE OF MANDELATE RACEMASE AT 2.5 ANGSTROMS RESOLUTION: IDENTIFICATION OF THE ACTIVE SITE AND POSSIBLE CATALYTIC RESIDUES

Mechanism of the reaction catalyzed by mandelate racemase. 2. Crystal structure of mandelate racemase at 2.5-A resolution: identification of the active site and possible catalytic residues.

Mandelate Racemase and Muconate Lactonizing Enzyme are Mechanistically Distinct and Structurally Homologous

EVLITGLRTRAVNVPLAYPVHTAVGTVGTAPLVLIDLATSAGVVGHSYLFAYTPVALKSLKQLLDDMAAMIVNEPLAPVS
LEAMLAKRFCLAGYTGLIRMAAAGIDMAAWDALGKVHETPLVKLLGANARPVQAYDSHSLDGVKLATERAVTAAELGFRA
VKTKIGYPALDQDLAVVRSIRQAVGDDFGIMVDYNQSLDVPAAIKRSQALQQEGVTWIEEPTLQHDYEGHQRIQSKLNVP
VQMGENWLGPEEMFKALSIGACRLAMPDAMKIGGVTGWIRASALAQQFGIPMSSHLFQEISAHLLAATPTAHWLERLDLA
GSVIEPTLTFEGGNAVIPDLPGVGIIWREKEIGKYLV

SHEET
BETA SHEETS ARE NAMED FOR THE MAJOR DOMAINS IN WHICH THEY
OCCUR: "N" FOR N-TERMINAL DOMAIN, "B" FOR BETA-BARREL
DOMAIN, AND "C" FOR C-TERMINAL DOMAIN. "F" REFERS TO THE
ACTIVE SITE FLAP. LIKEWISE, ALPHA HELICES ARE NAMED WITH
TWO CHARACTERS, THE FIRST REFERRING TO THE DOMAIN IN WHICH
THEY OCCUR.
THE SHEET PRESENTED AS *B* ON SHEET RECORDS BELOW IS
ACTUALLY AN EIGHT-STRANDED BETA-BARREL. THIS IS
REPRESENTED BY A NINE-STRANDED SHEET IN WHICH THE FIRST
AND LAST STRANDS ARE IDENTICAL.

MANDELATE RACEMASE ABSOLUTELY REQUIRES A DIVALENT METAL ION FOR ACTIVITY AND IS MOST ACTIVE WITH A MAGNESIUM (II) ION BOUND IN THE ACTIVE SITE. IN THIS CRYSTAL STRUCTURE, MANGANESE (II) WAS EXCHANGED INTO THE ACTIVE SITE IN ORDER TO FACILITATE X-RAY REFINEMENT OF THE POSITION OF THE BOUND METAL ION. MANGANESE (II) SUBSTITUTED MANDELATE RACEMASE IS ABOUT 48% AS ACTIVE AS HOLOENZYME CONTAINING MAGNESIUM (II); HOWEVER, VERY LITTLE STRUCTURAL CHANGE OF THE ENZYME IS APPARENT BETWEEN THESE FORMS. GIVEN THE MUCH HIGHER NATURAL ABUNDANCE OF MAGNESIUM (II) VS. MANGANESE (II), IT IS LIKELY THAT MAGNESIUM (II) IS THE PREDOMINANT COFACTOR FOR MANDELATE RACEMASE IN VIVO.

MSEVLITGLRTRAVNVPLAYPVHTAVGTVGTAPLVLIDLATSAGVVGHSYLFAYTPVALKSLKQLLDDMAAMIVNEPLAP
VSLEAMLAKRFCLAGYTGLIRMAAAGIDMAAWDALGKVHETPLVKLLGANARPVQAYDSHSLDGVKLATERAVTAAELGF
RAVKTKIGYPALDQDLAVVRSIRQAVGDDFGIMVDYNQSLDVPAAIKRSQALQQEGVTWIEEPTLQHDYEGHQRIQSKLN
VPVQMGENWLGPEEMFKALSIGACRLAMPDAMKIGGVTGWIRASALAQQFGIPMSSHLFQEISAHLLAATPTAHWLERLD
LAGSVIEPTLTFEGGNAVIPDLPGVGIIWREKEIGKYLV

ACID/BASE CATALYSTS RESPONSIBLE FOR PROTON ABSTRACTION AND REDELIVERY IN THE RACEMIZATION REACTION

THE BINDING SITE FOR THE CARBOXYL GROUP OF SUBSTRATE. THESE RESIDUES FUNCTION, ALONG WITH THE METAL ION, AS ELECTROPHILIC CATALYSTS