Step 1 Substrate dephosphorylation

Stabilization of the phosphoenzyme intermediate that is somewhere in between the other two cases. 

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The major relaxing transmitters are those that elevate the cAMP or cGMP concentration (Fig. 3). Adenosine stimulates the activity of cAMP kinase. The next step is not clear, but evidence has been accumulated that cAMP kinase decreases the calcium sensitivity of the contractile machinery. In vitro, cAMP kinase phosphorylated MLCK and decreased thereby the affinity of MLCK for calcium-calmodulin. However, this regulation does not occur in intact smooth muscle. Possible other substrate candidates for cAMP kinase are the heat stable protein HSP 20, (A heat stable protein of 20 kDa that is phosphorylated by cGMP kinase. It has been postulated that phospho-HSP 20 interferes with the interaction between actin and myosin allowing thereby smooth muscle relaxation without dephosphorylation of the rMLC.) Rho A and MLCP that are phosphorylated also by cGMP kinase I (Fig. 3). 

Unfortunately, the phosphorylated form of the starting aldehyde is expensive, and dephosphorylation by a phosphatase requires an additional step. Therefore, the challenge was to obtain a mutant aldolase that not only accepts nonphos-phorylated substrates but also turns over the enantiomeric aldehyde (29) stereoselectively with formation of (30), which is a precursor of carbohydrate (31) (see Scheme 2.8) [74] ... 

In order to account for the fact that almost all substrates are hydrolyzed at the same rate at high pH, even though dephosphorylation is not the sole rate determining step, Trentham and Gutfreund (98, 148) proposed that the mechanism involves a first-order rearrangement of the enzyme-substrate complex. This step is slow compared with the subsequent transfer of phosphate from substrate, S, to enzyme the final step being the liberation of phosphate from a phosphoryl enzyme intermediate ... 

Taking this result into account, we have developed an efficient enzymatic route catalyzed by FruA in which the acceptor substrate is a commercially available 2-haloacetaldehydes (chloro- or bromo-) 19 yielding 5-halo-D-xylulose after dephosphorylation catalyzed by acid phosphatase (see Section 18.2.3.1). The thiol was introduced after the enzymatic step by displacement of halogen with NaSH, to... 

Even prior to the elucidation of the first committed step of the riboflavin pathway, it had been shown that the benzenoid ring of riboflavin is assembled from two identical 4-carbon precursors. More specifically, the final step in the biosynthesis of the vitamin involves a dismutation of 6,7-dimethyl-8-ribityllumazine (6), where one of the substrate molecules serves as donor and the other as acceptor of a 4-carbon segment.19,20 6,7-Dimethyl-8-ribityllumazine, in turn, is formed in the penultimate step of the biosynthetic pathway from 5-amino-6-ribitylamino-4(3f/)-pyrimidinedione (3), an intermediate that is obtained from the product of GTP cyclohydrolase II by a sequence of deamination, side chain reduction, and dephosphorylation (Figure 3). The nature of the 4-carbon precursor required for the formation of 6,7-dimethyl-8-ribityllumazine (6) from 5-amino-6-ribitylamino-4(3f/)-pyrimidinedione (3) remained controversial for quite a long period, with working hypotheses including, but not limited to, tetroses, pentoses, and acetoin. 

Mammals produce sialic acid by aldolic condensation of phosphoenolpyruvate and Ai-acetylmannosamine 6-phosphate (reaction 12.1). A kinase enzyme catalyses the phosphorylation of A -acetylmannosamine and a phosphatase catalyses the hydrolysis of the phosphate of sialic acid. These phosphorylation and dephosphorylation steps are irreversible, such that the synthesis can be total even with low concentrations of the substrate. A variation of reaction (12.1), observed with the bacterium Neisseria meningitidis, uses non-phosphated /-acetylmannosamine. However, these were not the enzymes used in the preparative synthesis, which used instead a microbial aldolase which catalyses equilibrium (12.2). This enzyme probably plays a catabolic role in these organisms, but it functions in the synthetic sense in the presence of an excess of pyruvate. 

Despite the highly conserved structure of the PTP catalytic domain, PTPs have distinct substrate preferences from one another. For example, PTPIB and TCPTP preferentially dephosphorylate receptor tyrosine kinases and related adaptor molecules, whereas the KIM-family PTPs HePTP, STEP, and PTP-SL dephosphorylate specific MAP kinases. While the presence of distinct non-catalytic domains/motifs facilitate specific localization or binding to substrate proteins, PTP substrate specificity is also dictated by differences within the PTP catalytic domain itself. 

It is still unknown how the pyrimidine intermediate 5 is dephosphorylated (reaction VI). However, it is well established that the dephosphorylation product 6 is condensed with 3,4-dihydroxy-2-butanone 4-phosphate (8) by the catalytic action of lumazine synthase (reaction VIII). The carbohydrate substrate 8 is in turn obtained from ribulose phosphate (7) by a complex reaction sequence that is catalyzed by a single enzyme, 3,4-dihydroxy-2-butanone 4-phosphate synthase (reaction VII). As mentioned above, the lumazine 9 is converted to riboflavin (10) by the catalytic action of riboflavin synthase (reaction IX). Ultimately, riboflavin is converted to the coenzymes, riboflavin 5 -phosphate (flavin mononucleotide (FMN), 11) and flavin adenine dinucleotide (FAD, 12) by the catalytic action of riboflavin kinase (reaction X) and FAD synthase (reaction XI). These reaction steps are required in all organisms, irrespective of their acquisition of riboflavin from nutritional sources or by endogenous biosynthesis. 

Glycogen phosphorylase has been purified from adult A. suum muscle (3). The dephosphorylated enzyme, which requires AMP for activity, can be phosphorylated and activated by rabbit muscle phosphorylase kinase. Kinetic data and the observation that phosphorylase is not at equilibrium with its substrates and products, indicate that phosphorylase catalyzes the rate-limiting step of glycogenolysis in A. suum muscle. Phosphorylase activity correlates well with the rate of glycogenolysis observed in vivo... 

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