Creatine and Creatinine Metabolism - Physiological Reviews

1134 MARKUS WYSS AND RIMA KADDURAH-DAOUK Volume 80
triphosphates and even ADP are hydrolyzed by the enzyme.
In the presence of 1-methylhydantoin or dihydrouracil,
ATP and dATP display the lowest K m and highest
V max values. In the absence of an amide substrate, however,
ATP and dATP are not hydrolyzed at all, whereas
considerable hydrolytic activity was observed with the
other nucleoside triphosphates tested. These findings
point to a pronounced change in the specificity for nucleoside
triphosphates upon binding of an amide substrate
like 1-methylhydantoin or dihydrouracil.
The enzymatic activity of the 1-methylhydantoin
amidohydrolases critically depends on both divalent and
monovalent cations. EDTA almost completely abolishes
enzymatic activity. The potencies for reactivation of the
metal-free enzyme decrease in the order NH 4 � �
Rb � � K � � Cs � and Mg 2� � Mn 2� � Co 2� . 1-Methylhydantoin
amidohydrolases are also inhibited by sulfhydryl
and carbonyl group reagents, several metal ions, and
some other compounds.
In its catalytic properties, 1-methylhydantoin
amidohydrolase closely resembles two other amidehydrolyzing
enzymes, 5-oxoprolinase (EC 3.5.2.9) and
urea amidolyase (EC 3.5.1.45) (see Refs. 482, 714). All
three enzymes depend on ATP, Mg 2� or Mn 2� , and a
monovalent cation (NH 4 � or K � ) for catalysis. In analogy
to 1-methylhydantoin amidohydrolase, ATP hydrolysis
by 5-oxoprolinase is stimulated by L-2-imidazolidone-4-carboxylate
and dihydroorotate, which are not
hydrolyzed themselves by the enzyme. Therefore, it is
tempting to speculate that the three enzymes are evolutionarily
close.
In the case of anaerobic bacteria, evidence has been
provided for ATP-independent 1-methylhydantoin
amidohydrolases displaying K m values for 1-methylhydantoin
of 4.0–18.7 mM (278, 357). The enzyme from Tissierella
creatinini (see Ref. 335) had a pH optimum of
8.9. Its enzymatic activity critically depended on dithioerythritol
and was inhibited rather than stimulated by
NH 4 � and Mg 2� . In addition, a 20% higher rate of hydrolysis
was observed with hydantoin compared with 1-methylhydantoin.
If confirmed, these findings raise the question
whether ATP-dependent and ATP-independent
1-methylhydantoin amidohydrolases are characteristic of
aerobic and anaerobic bacteria, respectively, or whether
they represent adaptations to other physiological constraints.
N-carbamoylsarcosine amidohydrolase has been purified
from Arthrobacter, Micrococcus, and Moraxella
species (170) as well as from Pseudomonas putida 77
(486, 883). Moreover, the kinetic properties of the enzyme
have been characterized preliminarily in a cell-free extract
of Tissierella creatinini (278; see Ref. 335). For the
Pseudomonas enzyme, gel permeation chromatography
and ultracentrifugation experiments yielded a M r of
75,000–102,000, but an unusually high s 20,w of 13.9 S.
Because the subunit M r was determined to be 27,000 by
SDS-PAGE, the native molecules might be trimers, tetramers,
or even larger aggregates. The Arthrobacter enzyme
was shown by biochemical analysis, DNA sequencing, and
crystal structure determination to be a tetrameric molecule
composed of identical 264-amino acid subunits (819,
1156). Two subunits each form compact dimers which, in
turn, are held together in the tetramer by just a few
contacts. The four active sites are located at the intersubunit
interfaces of the compact dimers. Experiments with
reversible and irreversible inhibitors strongly suggest that
catalysis involves nucleophilic attack of N-carbamoylsarcosine
by the reactive thiol group of Cys-177, thereby
forming a covalent enzyme-thiohemiacetal intermediate.
Arg-202 is also likely to play a critical role, since it does
not only interact with the ligands but also blocks the entry
to the catalytic cleft. Arg-202 must move to allow exchange
of ligands.
Attempts to demonstrate the reversibility of the reaction
failed so far. The enzyme displays remarkable substrate
specificity: N-methyl-N-carbamoyl-DL-alanine, Ncarbamoylglycine,
N-carbamoyl-DL-alanine, and a variety
of other N-carbamoyl compounds were reported to be
hydrolyzed either slowly (�13% of the activity with Ncarbamoylsarcosine)
(486) or not at all (1156). Likewise,
these substances are, if at all, only weak inhibitors of
N-carbamoylsarcosine hydrolysis. Crn, Cr, sarcosine,
1-methylhydantoin, and hydantoin also do not serve as
substrates. N-carbamoylsarcosine amidohydrolase displays
stereospecificity, with the D- but not the L-isomers of
the N-carbamoyl compounds being hydrolyzed.
The K m for N-carbamoylsarcosine as well as the pH
optimum of the enzyme seem to depend on buffer composition,
with respective values of 0.125–7.1 mM and 7.0–
8.5. The V max of the purified enzyme at pH 7.5–8.0 and
25–37°C is on the order of 2 �mol N-carbamoylsarcosine
hydrolyzed � min �1 � (mg protein) �1 . N-carbamoylsarcosine
amidohydrolase is potently inhibited by thimerosal
(� merthiolate), p-chloromercuribenzoate, DTNB, Ag � ,
Hg 2� ,Cu 2� ,Zn 2� , and sodium arsenite, thus supporting
the reaction mechanism proposed above. Other metal
ions as well as 1,10-phenanthroline and 2,2�-bipyridine
had no significant effects on enzyme activity.
I. Sarcosine Oxidase, Sarcosine Dehydrogenase,
and Dimethylglycine Dehydrogenase
In various microorganisms, sarcosine is metabolized
further to glycine (8; for a review, see Ref. 958). In most
bacteria belonging to the genera Alcaligenes, Arthrobacter,
Bacillus, Corynebacterium, and Pseudomonas
as well as in fungi from the genera Cylindrocarpon
and Streptomyces, this degradation is achieved by a sarcosine
oxidase (EC 1.5.3.1; see Refs. 27, 500, 707, 803, 883,