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Members of the monoamine oxidase family of flavoproteins catalyze the oxidation of primary and secondary amines, polyamines, amino acids, and methylated lysine side chains in proteins. The enzymes have similar overall structures, with conserved flavin adenine dinucleotide (FAD)-binding domains and varied substrate-binding sites. Multiple mechanisms have been proposed for the catalytic reactions of these enzymes. The present review compares the structures of different members of the family and the various mechanistic proposals.

A combination of kinetic and structural approached is used for examining H373Q flavocytochrome [b.sub.2]. It is shown that the combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.

A study was done to assess the chemical mechanism of tryptophan 2-monooxygenase (TMO) turnover of L-alanine using pH studies, primary deuterium and [(15.super.N)] kinetic isotope effects. The outcomes ratified that the TMO oxidation of alanine happens through a hydride transfer mechanism.

The catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation are studied. The results suggest that all the aromatic amino acid hydroxylases share the same chemical mechanism but differ in the identity of the rate-limiting step.

The kinetics of reduction of ferric tyrosine hydroxylase (TyrH) by several reductants is determined by anaerobic stopped-flow spectrometry. The S40 TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of wild-type enzyme, indicating that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.

The 2.2 angstrom resolution crystal structure of nitroalkane oxidase trapped during the turnover of nitroethane as a covalent N5-FAD adduct (ES) is presented. The stability of N5-FAD adduct in NAO-ES is rationalized by structural features that include steric occlusion and hydrogen bonding that delocalizes the negative charge on NI of the isoalloxazine ring.

Research demonstrates that substitution of the serine40 residue of the tyrosine hydroxylase regulatory domain causes only minor changes in the steady-state kinetics of the enzyme. Results reveal that the serine hydroxyl is involved in the stabilization of the enzyme inhibited by catecholamine.

Research reveals that the C-H bond cleavage in the flavocytochrome b(sub)2 reaction does not involve lactate hydroxyl proton flight in the transition state as deduced from the measurement of primary deuterium and solvent kinetic isotope effects.

Seven residues in the active site of phenylalanine hydroxylase have been mutated to the corresponding residues in tyrosine hydroxylase. Side chains are not alike in the two enzymes, which are pterin dependent. It was found that reversing substrate specificities of phenylalanine and tyrosine hydroxylase aspartate 425 of tyrosine hydroxylase is necessary for L-DOPA formation.

Research has been conducted on Ser395 and Ser396. The use of site-directed mutagenesis to examine the role of these residues in the catalysis is discussed.

Research has been conducted on the nitroalkane oxidase that catalyzes nitroalkane oxidation to aldehydes or ketones. The investigation of the pH and kinetic isotope effects is described.

Research has been conducted on the flavoprotein nitroalkane oxidase. The kinetic mechanism of the active flavin adenine -containing enzyme form has been investigated.

Results demonstrate that nitroalkane oxidase contains a tyrosine residue in its active site and it may have a role in the substrate binding. Data discuss kinetic and structural characterization of the enzyme inactivated by tyrosine-specific tetranitromethane.

A study of the tyrosine hydroxylase activity regulation in the catecholamine neurotransmitters is presented. The decrease in affinity after phosphorylation has been investigated.

Position 5 of the indole ring of tryptophan appears to play an important role in the formation of 5-hydroxytryptophan from tryptophan catalyzed by tryptophan hydroxylase. Oxygen transfer to the amino acid seems to be the rate-limiting step in the reaction.

Research was conducted to purify and characterize the mutants of tyrosine hydroxylase (TyrH) with alanine substituted for Phe300 or Phe309. The structures of the catalytic domain of TyrH with and without dihydropterin bound have identified active-site residues that may have a role to play in the catalytic mechanism. Complementary evidence regarding the roles of various residues is provided by site-directed mutagenesis.

2-Oxo-3-pentynoate was found to be an active-site-directed inhibitor of certain flavoprotein oxidases. Tryptophan 2-monooxygenase is irreversibly inactivated in an active-site-directed manner. The addition of FAD provides no protection from inactivation, while the competitive inhibitor indole-3-acetamide fully protects the enzyme from inactivation. The rate of inactivation indicates saturation kinetics, consistent with the production of a reversible complex between the alkylating agent and the enzyme before inactivation occurs.

A study was conducted to analyze charged residues in the hydrophobic cleft of the active site of tyrosine hydroxylase. The 380B DNA synthesizer from Applied Biosystems was utilized to prepare the oligonucleotides. Plasmids were then introduced into Escherichia coli or BL2(DE3) cells to promote bacteria cell growth. Experimental results indicated that glutamate 332 promotes tetrahydropterin binding.

Several amino acids use tetrahydropterins as substrates. Iron is probably involved in the hydroxylating intermediate for the enzymes phenylalanine, tyrosine, and tryptophan.

A study was conducted to test whether mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase influences affinity for phenylalanine by characterizing a mutant protein of tyrosine hydroxylase. The Applied Biosystems model 380B DNA synthesizer was utilized to synthesize oligonucleotides. Results indicated that the stabilization of a radical and action as an active site base did not support a direct role in the hydroxyl moiety of tyrosine 371.

A study testing the hypothesis that phosphorylation induces catecholamines to bind to the ferric instead of the ferrous form of tyrosine hydroxylase is presented. Tyrosine hydroxylase is used to catalyze the biosynthesis of catecholamine neurotransmitters and hydroxylyze tyrosine to form dihydroxyphenylalanine. Results prove that catecholamines bind to the ferric form of tyrosine hydroxylase. This finding shows that the redox cycle between the ferric and ferrous forms of tyrosine hydroxylase is necessary.

A study was conducted on the oxygen-18 kinetic isotope effects for the tyrosine hydroxylase reaction to determine whether the activation of oxygen is the rate-limiting step for such reaction. A coupled substrate 4-methoxyphenylalanine and a totally uncoupled substrate 4-aminophenylalanine were used to measure the isotope effects. Results showed that the isotope effects are consistent with the bond order-to-oxygen change in the rate determining step.

A study was conducted to characterize the active flavin adenine dinucleotide (FAD)-containing structure of nitroalkane oxidase from Fusarium oxysporum. Dialysis was carried out to prepare the apoprotein of nitroalkane oxidase while the molecular mass of enzymes was determined by MALDI-TOF mass spectrometry. Results indicated no catalytic activity when FAD was replaced with 5-deaza-FAD. Findings also showed that the features of FAD in nitroalkane oxidase were within the range associated for other oxidases.

Tyrosine and phenylalanine hydroxylases have homologous catalytic domains and dissimilar regulatory domains. The influence of the regulatory domains on the substrate specificities was determined by producing truncated and chimeric mutants of tyrosine and phenylalanine hydroxylase. The truncated proteins had low binding specificity for either amino acid. Attachment of either regulatory domain substantially enhanced the specificity, but the specificity was determined by the catalytic domain in the chimeric proteins.

A new strategy for hydroxylating tyrosine to dihydoxyphenylalanine is discussed. It involves using 4-substituted phenylalanines as substrate for the iron-containing enzyme tyrosine hydroxylase. The results showed formation of multiple hydroxylated products, with a different site of hydroxylation for every product. In general, the site of hydroxylation shifted from the 4- to the 3-position of the aromatic ring as the size of the substituent increased.

Analysis of the pH and kinetic isotope effects on steady-state kinetic parameters of the flavoprotein tryptophan 2-monooxygenase suggests that the pH and isotope effects agree with a large forward commitment for tryptophan as substrate for the enzyme. The solvent isotope effect on the Vmax value with tryptophan is 2.5, agreeing with an association between slow proton transfer and product release.

Kinetic studies of the catalysis by flavoprotein tryptophan 2-monooxygenase of the oxidative decarboxylation of tryptophan to indole-3-acetamide, carbon dioxide and water suggest that a rapid phase of the mechanism is followed by a slow, concentration and isotope-independent phase that is associated with the dissociation of a reduced enzyme-imino acid complex. The rate of indole-3-acetamide formation is comparable with the rate of reaction with oxygen, followed by product dissociation.