Glycerol 3-Phosphate Dehydrogenase Catalyzed Hydride Transfer: Enzyme Activation by Cofactor Pieces.

Glycerol 3-phosphate dehydrogenase catalyzes reversible hydride transfer from glycerol 3-phosphate (G3P) to NAD+ to form dihydroxyacetone phosphate; from the truncated substrate ethylene glycol to NAD+ in a reaction activated by the phosphite dianion substrate fragment; and from G3P to the truncated nicotinamide riboside cofactor in a reaction activated by adenosine 5'-diphosphate, adenosine 5'-monophosphate, and ribose 5-phosphate cofactor fragments. The sum of the stabilization of the transition state for GPDH-catalyzed hydride transfer reactions of the whole substrates by the phosphodianion fragment of G3P and the ADP fragment of NAD+ is 25 kcal/mol. Fourteen kcal/mol of this transition state stabilization is recovered as phosphite dianion and AMP activation of the reactions of the substrate and cofactor fragments. X-ray crystal structures for unliganded GPDH, for a binary GPDH·NAD+ complex, and for a nonproductive ternary GPDH·NAD+·DHAP complex show that the ligand binding energy is utilized to drive an extensive protein conformational change that creates a caged complex for these ligands. The phosphite dianion and AMP fragments are proposed to activate GPDH for the catalysis of hydride transfer by stabilization of this active caged complex. The closure of a conserved loop [292-LNGQKL-297] during substrate binding stabilizes the G3P and NAD+ complexes by interactions, respectively, with the Q295 and K296 loop side chains. The appearance and apparent conservation of two side chains that interact with the hydride donor and acceptor to stabilize the active closed enzyme are proposed to represent a significant improvement in the catalytic performance of GPDH.

Glycerol 3-Phosphate Dehydrogenase: Role of the Protein Conformational Change in Activation of a Readily Reversible Enzyme-Catalyzed Hydride Transfer Reaction.

Kinetic parameters are reported for glycerol 3-phosphate dehydrogenase (GPDH)-catalyzed hydride transfer from the whole substrate glycerol 3-phosphate (G3P) or truncated substrate ethylene glycol (EtG) to NAD, and for activation of the hydride transfer reaction of EtG by phosphite dianion. These kinetic parameters were combined with parameters for enzyme-catalyzed hydride transfer in the microscopic reverse direction to give the reaction equilibrium constants Keq. Hydride transfer from G3P is favored in comparison to EtG because the carbonyl product of the former reaction is stabilized by hyperconjugative electron donation from the -CH2R keto substituent. The kinetic data show that the phosphite dianion provides the same 7.6 ± 0.1 kcal/mol stabilization of the transition states for enzyme-catalyzed reactions in the forward [reduction of NAD by EtG] and reverse [oxidation of NADH by glycolaldehyde] directions. The experimental evidence that supports a role for phosphite dianion in stabilizing the active closed form of the GPDH (EC) relative to the ca. 6 kcal/mol more unstable open form (EO) is summarized.

Triosephosphate Isomerase: The Crippling Effect of the P168A/I172A Substitution at the Heart of an Enzyme Active Site.

The P168 and I172 side chains sit at the heart of the active site of triosephosphate isomerase (TIM) and play important roles in the catalysis of the isomerization reaction. The phosphodianion of substrate glyceraldehyde 3-phosphate (GAP) drives a conformational change at the TIM that creates a steric interaction with the P168 side chain that is relieved by the movement of P168 that carries the basic E167 side chain into a clamp that consists of the hydrophobic I172 and L232 side chains. The P168A/I172A substitution at TIM from Trypanosoma brucei brucei (TbbTIM) causes a large 120,000-fold decrease in kcat for isomerization of GAP that eliminates most of the difference in the reactivity of TIM compared to the small amine base quinuclidinone for deprotonation of catalyst-bound GAP. The I172A substitution causes a > 2-unit decrease in the pKa of the E167 carboxylic acid in a complex to the intermediate analog PGA, but the P168A substitution at the I172A variant has no further effect on this pKa. The P168A/I172A substitutions cause a 5-fold decrease in Km for the isomerization of GAP from a 0.9 kcal/mol stabilization of the substrate Michaelis complexes. The results show that the P168 and I172 side chains play a dual role in destabilizing the ground-state Michaelis complex to GAP and in promoting stabilization of the transition state for substrate isomerization. This is consistent with an important role for these side chains in an induced fit reaction mechanism [Richard, J. P. (2022) Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution. Biochemistry 61, 1533-1542].

The Role of Asn11 in Catalysis by Triosephosphate Isomerase.

Four catalytic amino acids at triosephosphate isomerase (TIM) are highly conserved: N11, K13, H95, and E167. Asparagine 11 is the last of these to be characterized in mutagenesis studies. The ND2 side chain atom of N11 is hydrogen bonded to the O-1 hydroxyl of enzyme-bound dihydroxyacetone phosphate (DHAP), and it sits in an extended chain of hydrogen-bonded side chains that includes T75' from the second subunit. The N11A variants of wild-type TIM from Trypanosoma brucei brucei (TbbTIM) and Leishmania mexicana (LmTIM) undergo dissociation from the dimer to monomer under our assay conditions. Values of Kas = 8 × 103 and 1 × 106 M-1, respectively, were determined for the conversion of monomeric N11A TbbTIM and LmTIM into their homodimers. The N11A substitution at the variant of LmTIM previously stabilized by the E65Q substitution gives the N11A/E65Q variant that is stable to dissociation under our assay conditions. The X-ray crystal structure of N11A/E65Q LmTIM shows an active site that is essentially superimposable on that for wild-type TbbTIM, which also has a glutamine at position 65. A comparison of the kinetic parameters for E65Q LmTIM and N11A/E65Q LmTIM-catalyzed reactions of (R)-glyceraldehyde 3-phosphate (GAP) and (DHAP) shows that the N11A substitution results in a (13-14)-fold decrease in kcat/Km for substrate isomerization and a similar decrease in kcat for DHAP but only a 2-fold decrease in kcat for GAP.

Effect of Whole-Body Vibration Exercise on Hamstrings-to-Quadriceps Ratio, Walking Performance, and Postural Control in Children With Hemiparetic Cerebral Palsy: A Randomized Controlled Trial.

OBJECTIVE: The purpose of this study was to investigate the effect of whole-body vibration (WBV) exercises combined with traditional physiotherapy on the hamstrings-to-quadriceps (H:Q) ratio, walking ability, and control of posture in children with hemiparetic cerebral palsy (CP). METHODS: A total of 34 children with spastic hemiparetic CP (boys and girls) participated in this 2-arm, parallel, randomized controlled trial. The inclusion criteria were spasticity ranging from 1 to 1+, gross level skills (I and II), at least 1 meter tall, standing alone, and walking forward and backward. They were randomly allocated to the control group (traditional physiotherapy) and study group and were treated by the same physiotherapy program combined with WBV training (3 times per week for 2 successive months). Quadriceps and hamstring muscle strength, walking performance, and postural control were evaluated before and after intervention by a blinded assessor. RESULTS: The post-intervention values of the hamstring and quadriceps muscle force, gross motor function, and stability indices of the 2 groups were higher than the pre-values (P < .05). In addition, the post-values of the study group were higher than those of the control group (P < .05). Regarding the H:Q ratio, there was no significant difference between pre-values or the post-values of both groups (P = .948 and P = .397, respectively). There were no significant differences between the pre- and post-values of each group (P = .500 and P = .195, respectively). CONCLUSION: Eight weeks of WBV training combined with traditional physiotherapy was more effective than traditional physiotherapy alone in improving walking ability and postural control. Furthermore, the combined intervention strengthened the quadriceps and hamstring muscles, with no change in the H:Q ratio in children with hemiparetic CP.

Computed tomographic evaluation of C5 root exit foramen in patients with cervical spondylotic myelopathy.

BACKGROUND: Narrowing of the intervertebral foramen for C5 root and a larger superior articular process in myelopathic patients with postlaminoplasty motor dominant C5 radiculopathy has been reported. We investigated whether the C4-5 foraminal dimensions and surface area in patients with cervical spondylotic myelopathy are universally smaller than the intervertebral foramina at other cervical levels. METHODS: The study population consisted of 44 consecutive patients (sex: 24 males and 20 females), averaging 55.7 years of age (range 42-84) years who presented with clinical features suggestive of cervical spondylotic myelopathy. Using computed tomography (CT) imaging, we prospectively compared height, transverse diameter, and surface area of the C4-5 foramen to those of C3-4, C5-6 and C6-7 foramina of the same side in the whole study population as well as in male and female patients. RESULTS: In the whole study population at C4-5 intervertebral foramen the mean foraminal height was 8.37 ± 1.3 mm on the right and 8.85 ± 1.16 mm on the left; and the mean foraminal transverse diameter on the right was 4.97 ± 1.35 mm and 5.14 ± 1.16 mm on the left. No statistically significant difference was found between the measurements in the whole study population at various levels, between or within male and female patient groups. CONCLUSION: C4-5 intervertebral foramen is not uniformly smaller in patients with cervical spondylotic myelopathy.

Blake's pouch cyst and Werdnig-Hoffmann disease: Report of a new association and review of the literature.

BACKGROUND: We report a case of a neonate with proximal spinal muscular atrophy (SMA) type 1 (also known as Werdnig-Hoffmann disease or severe infantile acute SMA) associated with a Blake's pouch cyst; a malformation that is currently classified within the spectrum of Dandy-Walker complex. The association of the two conditions has not been previously reported in the English literature. A comprehensive review of the pertinent literature is presented. CASE DESCRIPTION: A male neonate was noted to have paucity of movement of the four limbs with difficulty of breathing and poor feeding soon after birth. Respiratory distress with tachypnea, necessitated endotracheal intubation and mechanical ventilation. Pregnancy was uneventful except for decreased fetal movements reported by the mother during the third trimester. Neurological examination revealed generalized hypotonia with decreased muscle power of all limbs, nonelicitable deep tendon jerks, and occasional tongue fasciculations. Molecular genetic evaluation revealed a homozygous deletion of both exons 7 and 8 of the survival motor neuron 1 (SMN1) gene, and exon 5 of the neuronal apoptosis inhibitory protein (NAIP) gene on the long arm of chromosome 5 consistent with Werdnig-Hoffmann disease (SMA type 1). At the age of 5 months, a full anterior fontanelle and abnormal increase of the occipito-frontal circumference were noted. Computed tomographic (CT) scan and magnetic resonance imaging (MRI) of the brain revealed a tetraventricular hydrocephalus and features of Blake's pouch cyst of the fourth ventricle. CONCLUSIONS: This case represents a previously unreported association of Blake's pouch cyst and SMA type 1.

Computed tomographic evaluation of the distance between the medial border of longus colli muscle and foramen transversarium in ventral approaches to the subaxial cervical spine.

AIM: Iatrogenic vertebral artery (VA) injury during ventral approaches to the subaxial cervical spine ranges from 0.22% to 2.77%. Evaluation of the extent of safe lateral working distance before the V2 segment of the VA is reached can be helpful to avoid this complication. MATERIAL AND METHODS: In 100 patients (48 males and 52 females) axial computed tomographic scanning was used to measure the distance from the medial border of longus colli muscle (LCM) to the medial border of the foramen transversarium along the anterior border of the vertebral body at each level from C3-4 down to C6-7. The arithmetic mean of the 2 measurements at the upper and lower end-plates of the corresponding level was considered representative of the safe lateral working distance at this level. Statistical significance was set as P value < 0.001. RESULTS: No statistically significant difference was found between the measurements in the whole study population at various levels or between subgroups. A gradual increase in the distances was noticed from C3-4 down to C6-7 level in all subgroups except for spondylotic males. CONCLUSION: This study offers useful morphometric data that can help the surgeon avoid VA injury during anterior procedures to the subaxial cervical spine.